- Volume 141, Issue 5, 1995
Volume 141, Issue 5, 1995
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Anaerobic metabolism in Bacillus licheniformis NCIB 6346
More LessSUMMARYThe products of anaerobic metabolism of glucose and its derivatives sorbitol, gluconate and glucuronate by Bacillus licheniformis have been determined by proton NMR. Glucose was fermented through mixed-acid fermentation pathways to acetate, 2,3-butanediol, ethanol, formate, lactate, succinate and pyruvate. However, the bacterium was incapable of fermenting the three glucose derivatives. When B. licheniformis cells were incubated anaerobically with glucose in the presence of nitrate, the reduced products and formate did not appear and acetate was formed as the major metabolite. Growth and formation of acetate was also observed when B. licheniformis cells were incubated anaerobically with each of the three glucose derivatives, in the presence of nitrate. A formate-nitrate oxido-reductase system was induced under anaerobic conditions, with increased activities when nitrate was added to the anaerobic growth medium. However no activity was detected when cell; were grown in the presence of molecular oxygen. Formate-nitrate oxido-reductase activity was absent in chlorate-resistant mutants isolated spontaneously or following Tn917 insertional mutagenesis. The spontaneous mutants fermented glucose in the presence of nitrate suggesting that they were incapable of nitrate respiration, due to a deficiency in one or more components of the formate-nitrate oxido-reductase system. Two insertional mutants exhibited elevated β-galactosidase activity when grown in the presence of nitrate.
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Zinc- and arsenic-resistant strains of Thiobacillus ferrooxidans have increased copy numbers of chromosomal resistance genes
More LessSUMMARYPulsed-field gel electrophoresis (PFGE) was used to examine chromosomal DNA from various strains of Thiobacillus ferrooxidans: these were the reference strain VKM-458, strains isolated from different environments and pilot plants for processing gold-bearing concentrates, and strains experimentally adapted to high zinc and arsenic concentrations in growth medium. The restriction endonuclease Xbal digested T. ferrooxidans VKM-458 chromosomal DNA into a number of fragments sufficient for identification of their size and calculation of the size of the entire genome (2855/pm44 kb). Restriction fragment length polymorphism of the chromosomal DNA in various strains suggests the usefulness of this approach for analysis of the diversity of T. ferrooxidans strains and for the study of strain stability under conditions of industrial utilization. A comparison of Xbal-restriction patterns in parent strains and in strains with acquired enhanced resistance to zinc or arsenic revealed amplification of certain fragments in the resistant strains, i.e. a 98 kb fragment in strain TFZ and a 28 kb fragment in strain VKM-458As2. We suggest that the enhanced resistance to toxic metals in T. ferrooxidans is gained through increase of the copy number of resistance genes and enhanced synthesis of proteins involved in resistance.
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Organization of carbamoyl-phosphate synthase genes in Neisseria gonorrhoeae includes a large, variable intergenic sequence which is also present in other Neisseria species
More LessSUMMARYThe carbamoyl-phosphate synthase (CPS) enzyme in prokaryotes is a heterodimer, encoded by genes commonly called carA and carB. In most prokaryotes examined, these genes are separated by up to 24 bp and are cotranscribed. In Pseudomonas aeruginosa, carA and carB are also co-transcribed, but are separated by 682 bp. We have determined the complete DNA sequence of the carA and carB genes of Neisseria gonorrhoeae strain CH811. carA (1125 bp) and carB (3237 bp) are similar in size and sequence to other prokaryotic CPS genes, however they are separated by an intervening sequence of 3290 bp which has no similarity to the intervening sequence between other CPS genes; furthermore, putative transcription terminators are found downstream of both carA and carB. Several neisserial repetitive sequences were identified within the 9 kb sequenced, as well as novel 120 and 150 bp repeats (designated RS6 and RS7, respectively) which were found within the intervening sequence between carA and carB. To determine whether the intervening sequence observed in N. gonorrhoeae CH811 was not unusual, the sequence between carA and carB was amplified by PCR from 30 isolates of N. gonorrhoeae. The intervening sequence was found to vary in size, from approximately 2·2 to 3·7 kb, although the carA and carB genes themselves did not vary in size in isolates with functional CPS enzyme. A similar large, variably sized intervening sequence was also found between the carA and carB genes of 12 isolates of N. meningitidis and 18 commensal Neisseria isolates comprising nine species. This unexpected organization of the CPS genes in N. gonorrhoeae is therefore widespread throughout the genus Neisseria.
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The Bacillus subtilis dnal gene is part of the dnaB operon
More LessSUMMARYThe dnal gene of Bacillus subtilis, previously identified through the isolation of the dnal2 mutant, was found to be the second gene of the dnaB operon. The nucleotide substitution in the dnal2 mutant gene was determined.
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Crystalline surface protein of Peptostreptococcus anaerobius
More LessSUMMARYThe surface ultrastructure of three anaerobic Gram-positive cocci frequently encountered in oral infections, Peptostreptococcus micros, P. magnus and P. anaerobius, was studied. The type strains of P. micros (DSM 20468) and P. anaerobius (ATCC 27337), several clinical isolates of both species and the type strain of P. magnus (DSM 20470) were included. Thin-sectioned cells studied by electron microscopy revealed a homogeneous layer outside the peptidoglycan layer in P. anaerobius. In P. micros and P. magnus a more amorphous layer was present. No periodic structures were seen in negatively stained whole cells of these three species. However, in freeze-etched cells of P. anaerobius a crystalline surface protein layer (S-layer) was detected. No periodicity was seen in any of the P. micros strains or the magnus type strain by the methods used, but a periodic pattern was observed in negatively stained specimens of cell wall fragments of sonicated P. anaerobius cells. No capsular material was visible outside the S-layer in P. anaerobius. The cells of the Peptostreptococcus spp. were extracted for 30 min with detergents and urea. One er cent SDS and 6 M urea both extracted a major 78 kDa protein from all strains of P. anaerobius. Extraction of P. micros and P. magnus cells did not reveal any major protein bands comparable to that of P. anaerobius. Surface biotinylation of cells followed by Western blotting and detection by alkaline-phosphatase-conjugated extravidin showed strong staining of the 78 kDa band in P. anaerobius, further indicating that this molecule is located on the surface of the cell and is the S-protein. Another protein, of 127 kDa, was also detected by surface biotinylation in all P. anaerobius strains. No differences were detected in colony morphology or cell surface structures between the four strains. Isoelectric focusing of the proteins of P. anaerobius revealed that the S-proteins of individual strains have different pl values, ranging from 5.3 to 6.9.
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Cellular fatty acyl and alkenyl residues in Megasphaera and Pectinatus species: contrasting profiles and detection of beer spoilage
More LessSUMMARYThe strictly anaerobic Gram-negative beer spoilage bacteria Megasphaera cerevisiae, Pectinatus cerevisiiphilus and P. frisingensis were subjected to cellular fatty acid analysis, employing acid- and base-catalysed cleavage, gas chromatography and mass spectrometry. M. cerevisiae contained 12:0, 16:0, 16:1, 18:1, 17:cyc, 19:cyc, 12:0(3OH), 14:0(3OH) as the main fatty acids, and alk-1-enyl chains instead of acyl chains were detected to a considerable extent (14% of total fatty acids), indicating the presence of plasmalogens. The fatty acid pattern of M. cerevisiae was almost identical to that of M. elsdenii, the only species previously assigned to this genus. P. cerevisiiphilus and P. frisingensis yielded fatty acids that were heavily dominated by odd-numbered chains; 11:0, 15:0, 17:1, 18:cyc and 13:0(3OH) were the main fatty acids detected in both species. Alk-1-enyl chains with similar chain lengths were also found. Both Pectinatus species contained six different 3-hydroxy fatty acids with chain lengths between 11 and 15 carbons, 13:0(3OH) being dominant and the others accounting for generally less than 1% of total fatty acids. Among the minor components, an unsaturated 3-hydroxy fatty acid was detected which was shown to be 13:1(30H). In addition, fatty acid analysis was shown to be applicable to detection of bacterial contamination of beer.
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An inner cell wall protein (cwp1) from conidia of the entomopathogenic fungus Beauveria bassiana
More LessSUMMARYFollowing the removal of the rodlet layer from aerial or submerged conidia of the entomopathogenic deuteromycetous fungus Beauveria bassiana, SDS-insoluble, formic-acid-extractable proteins were found in the residual cell wall material. Two major proteins (12·8 and 14·0 kDa) were extracted with formic acid from fractured aerial and submerged conidia but not from blastospores. Oxidation of the sample extracted by formic acid resulted in a single protein band (15·4 kDa) as judged by SDS-PAGE. Antibodies against this cell wall protein (cwp1) did not cross-react with cell wall extracts from the entomopathogenic deuteromycetous fungi Verticillium lecanii or Metarhizium anisopliae. Western blot analysis of two-dimensional gels revealed at least three acidic isoforms (pl 4·0-4·8) of cwp1. Immunohistological studies revealed that the cwp1 was primarily localized in the cell wall of aerial and submerged conidia but not in blastospores, Immunolocalization was possible only if the conidia were previously boiled in 5% (v/v) β-mercaptoethanol. The N-terminal sequence of cwp1 showed no similarities with other published sequences. Our results suggest that at least two major species of SDS-insoluble, formic-acid-extractable proteins exist in cell walls of B. bassiana aerial or submerged conidia; one is the hydrophobin which occurs in the outermost rodlet layer and the other, cwp1, occurs primarily next to the rodlet layer.
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The tylosin producer, Streptomyces fradiae, contains a second valine dehydrogenase
More LessSUMMARYA second NAD-dependent valine dehydrogenase (VDH) of Streptomyces fradiae was detected and purified to homogeneity by affinity chromatography on Reactive-Blue 2 Sepharose followed by gel filtration and Mono Q fast protein liquid chromatography. The relative molecular masses of the native enzyme and its subunits were determined to be 80000 and 41000, respectively, indicating that the enzyme is a homodimer. The enzyme was the only active VDH in S. fradiae; its activity was significantly induced by L-valine, but was repressed by ammonia. Among branched- and straight-chain amino acids that serve as enzyme substrates, L-2-aminobutyrate and L-valine are preferred. Significant activities were found with deamino-NAD+ and 3-pyridinealdehyde-NAD-. The molecular and catalytic properties of the enzyme distinguish it from the enzyme previously purified, and thus indirectly indicate the existence of two VDHs in S. fradiae.
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Typing of Staphylococcus aureus strains by PCR-amplification of variable-length 16S-23S rDNA spacer regions: characterization of spacer sequences
More LessSUMMARYTo develop a rapid and accurate method of typing large numbers of clinical isolates of Staphylococcus aureus, the spacer region C of the rRNA operon [1391-507 (16S-23S)] was enzymically amplified from 322 strains. When the products were separated by denaturing PAGE, 15 variable-length rrn alleles were demonstrated, ranging in size from 906 to 1223 bp. The variable-length HpaII-digested region C [(region E; 1446-196 (16S-23S)] amplification products were cloned into M13mp18RF to sequence separate variable-length alleles. A total of 17 region E inserts were sequenced, aligned and divided into nine alleles by length (938-1174) and sequence properties. The 16S-23S spacer rDNA varied in length (303-551 bp) and in properties; three alleles contained a tRNAlle gene alone, two alleles contained a tRNAlle and a tRNAAla gene, and four alleles lacked tRNA genes. The sequences of two alleles showed less than 1% variation when isolated from two or three S. aureus strains. The 48 penicillin-and methicillin-sensitive strains were divided into 26 ribotypes; in contrast, the 274 methicillin-resistant S. aureus (MRSA) strains were divided into nine ribotypes (A-I) with 97% typing as either ribotype A or B (rrnL was missing in B). The sequence conservation of the rrn operons argues for the use of the 16S-23S spacer region as a stable and direct indicator of the evolutionary divergence of S. aureus strains.
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Nitrogen-regulated transcription and enzyme activities in continuous cultures of Saccharomyces cerevisiae
SUMMARYVariations in the transcription of nitrogen-regulated genes and in the activities of nitrogen-regulated enzymes of the yeast Saccharomyces cerevisiae were studied by changing the carbon and nitrogen fluxes. S. cerevisiae was grown in continuous culture at various dilution rates (D) under nitrogen limitation with NH4Cl as sole nitrogen source. With an increase in D from 0.05 to 0.29 h-1, both the glucose and the ammonia flux increased sixfold. The activities of the two ammonia-incorporating enzymes, NADPH-dependent glutamate dehydrogenase (NADPH-GDH) and glutamine synthetase (GS), encoded by GDH1 and GLN1, respectively, increased with increasing D, while the activity of the glutamate-degrading enzyme, NAD-dependent glutamate dehydrogenase (NAD-GDH), decreased. Surprisingly, no changes were observed in the transcription of GDH1 and GLN1; however increased D was accompanied by an increase in GAP1 transcription. At the metabolite level, the increase in the glucose and nitrogen flux did not result in changes in the intracellular 2-oxoglutarate, glutamate or glutamine concentrations. It is shown that growth on ammonia alone is not sufficient to cause repression of GAP1 and GLN1 transcription and that the regulation of GAP1 transcription and both NADPH-GDH and GS activity is not an on/off switch, but is gradually modulated in correlation with the ammonia concentration.
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Mutations in the glycerol kinase gene restore the ability of a ptsGHI mutant of Bacillus subtilis to grow on glycerol
More LessSUMMARYAlthough glycerol is not taken up via the phosphotransferase system (PTS) in Bacillus subtilis, some mutations that affect the general components of the PTS impair the ability of cells to grow on glycerol. Five revertants of a pts deletion mutant that grow on glycerol were analysed. They were shown to carry mutations in the glycerol kinase gene. These are missense mutations located in parts of the glpK gene that could encode regions important for the activity of glycerol kinase. The results strongly suggest that the main effect of the PTS on glycerol utilization in B. subtilis is mediated via glycerol kinase.
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Evolution of the korA-oriV segment of promiscuous IncP plasmids
More LessSUMMARYPlasmids belonging to Escherichia coli incompatibility group P are of particular interest because they can transfer between, and be stably maintained in, almost all Gram-negative bacterial species. The segment of the IncPα plasmid genome between the key regulatory gene korA and the vegetative replication origin, oriV, encodes a series of operons co-regulated with replication and transfer functions by the KorA protein. To determine which of these genes are likely to have an important role in IncP plasmid survival the equivalent region of the distantly related IncPβ plasmid R751 was sequenced. Sequence comparisons show that the kla operon (formerly the kilA locus, which is also responsible for a cryptic tellurite-resistance determinant) is completely absent from R751. Similarly in the kle region, which encodes genes associated with the KilE+ phenotype of unknown function, kleC and kleD, which we proposed arose by a duplication of kleA and kleB, are also completely absent. The genes that are conserved are klcA (formerly kilC, responsible for the KilC+, and recently proposed to be involved in overcoming restriction barriers during transfer), klcB (an ORF interrupted by Tn 1 insertion in RK2), korC (a transcriptional repressor which controls the kleK and kle operons), and kleA, kleB, kleE and kleF. A striking feature of the organization in R751 is the lack of the strong transcriptional termination signals which are present in IncPα plasmids. The degree of divergence between the plasmids facilitates the identification of motifs of probable functional importance in the primary protein sequences.
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Cloning of a Candida albicans peptide transport gene
SUMMARYA Candida albicans peptide transport gene, CaPTR2, was cloned from a C. albicans genomic library by functional complementation of a peptide transport deficient mutant (strain ptr2-2) of Saccharomyces cerevisiae. CaPTR2 restored peptide transport to transformants as determined by uptake of radiolabelled dileucine, growth on dipeptides as sources of required amino acids, and restoration of growth inhibition by toxic peptides. Plasmid curing experiments demonstrated that the peptide transport phenotype was plasmid borne. CaPTR2 was localized to chromosome R of C. albicans by contour-clamped homologous electric field gel chromosome blots. Deletion subclones and frameshift mutagenesis were used to narrow the peptide transport complementing region to a 5:1 kb DNA fragment. DNA sequencing of the complementing region identified an ORF of 1869 bp containing an 84 nucleotide intron. The deduced amino acid sequence predicts a protein of 70 kDa consisting of 623 amino acids with 12 hydrophobic segments. A high level of identity was found between the predicted protein and peptide transport proteins of S. cerevisiae and Arabidopsis thaliana. This study represents the first steps in the genetic characterization of peptide transport in C. albicans and initiates a molecular approach for the study of drug delivery against this pathogen.
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The immunogenicity of recombinant Mycobacterium smegmatis bearing BCG genes
More LessSUMMARYSpecific pathogen-free C57BL/6 mice infected with recombinant Mycobacterium smegmatis (rM. smegmatis) bearing BCG genes showed increased splenic survival compared to those receiving the vector control (plasmid DNA only). The mouse-passaged rM. smegmatis (J3R) survived in peritoneal macrophages better than the vector control, regardless of whether the macrophages were infected in vivo or in vitro. When rM. smegmatis J3R was cultured in synthetic Proskauer-Beck-Tween medium, protein bands characteristic of BCG culture filtrates and not present in the vector control preparation were observed. Mice immunized with two doses of heat-killed J3R suspended in Freund's adjuvant were able to limit the growth of virulent Mycobacterium tuberculosis within the lung and spleen compared to that observed in control mice receiving adjuvanted vector control or Freund's adjuvant alone.
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Division-inhibition capacity of penicillin in Escherichia coli is growth-rate dependent
More LessSUMMARYGrowing bacteria are sensitive to various -lactam derivatives due to their interference with peptidogly can biosynthesis. At low concentrations, penicillin G (benzylpenicillin) blocks cell division without affecting mass growth rate. The MIC for division of Escherichia coli B/r (H266) was found to depend on the growth rate, which was modified by the nutritional conditions. Our hypothesis, that division sensitivity is proportional to the rate of peptidoglycan synthesis for septum formation, as well as to cell circumference, was thus confirmed.
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Ammonia-dependent synthesis and metabolic channelling of carbamoyl phosphate in the hyperthermophilic archaeon Pyrococcus furiosus
More LessSUMMARYThe biosynthesis of carbamoyl phosphate (CP), a metabolic precursor of arginine and the pyrimidines was investigated in the hyperthermophilic archaeon Pyrococcus furiosus. The half-life of CP was found to be less than 2 s in the optimum temperature range of this organism (100-102 °C). The carbamoyl-phosphate synthase (CPSase) of P. furiosus uses ammonia as the nitrogen donor, and not glutamine like all micro-organisms investigated so far. The Mr of the enzyme, which is devoid of regulatory properties, is 70000, at variance with that of known CPSases. The possible significance of these findings with regard to hyperthermophilic nitrogen metabolism is discussed. Competition experiments with P. furiosus crude extracts indicated a marked preference of ornithine carbamoyltransferase (OTCase) for CP synthesized by CPSase rather than for CP added to the reaction mixture. In addition, the bisubstrate analogue -N-phosphonoacetyl-L-ornithine inhibits the formation of citrulline from bicarbonate, ammonia, ATP and ornithine much less than its synthesis from ornithine and CP in the presence of free OTCase. Such results suggest that, in vivo, CPSase and OTCase associate in a complex able to channel CP. Such a channelling may confer protection to CP, thus avoiding the accumulation of toxic amounts of cyanate arising from its decomposition as well as the waste of the two molecules of ATP required for its synthesis.
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An operon encoding aspartokinase and purine phosphoribosyltransferase in Thermus flavus
More LessSUMMARYThe nucleotide sequence of a 1:1 kb Xhol-HindIII fragment downstream of the malate dehydrogenase (mdh) gene of Thermus flavus revealed the presence of an ORF and an incomplete ORF lacking its NH2-terminal portion, in the opposite orientation to that of the mdh gene. These two genes overlapped with each other, sharing two base pairs, suggesting that these genes are co-transcribed in a single mRNA. One ORF (termed gpt) encoded a protein of 154 amino acids showing significant amino acid sequence similarity to purine phosphoribosyltransferases, such as xanthine-guanine phosphoribosyltransferase of Escherichia coli and human hypoxanthine phosphoribosyltransferase. Cloning and sequencing of the upstream region of the gpt gene, together with sequence comparison of the gene product encoded by the region upstream of gpt, suggested that the upstream ORF encoded two in-frame overlapping aspartokinase genes, askA, encoding the β-subunit of 405 amino acids, and askB, encoding the β-subunit of 161 amino acids, which was part of the 3′ portion of askA. Consistent with the sequence data, the askAB and the gpt genes conferred the heat-stable enzyme activities of aspartokinase and phosphoribosyltransferase, respectively, on E. coli. Preliminary characterization of these enzymes produced in E. coli is described.
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Involvement of ArgR and PepA in the pairing of ColE1 dimer resolution sites
More LessSUMMARYDimer formation and associated copy number depression is an important cause of multicopy plasmid instability. Natural multicopy plasmids employ site-specific recombination to convert dimers to monomers, thus maximizing the number of independently segregating molecules at cell division. Resolution of dimers of Escherichia coli plasmid ColE1 requires the plasmid cer site and at least four chromosome-encoded proteins: the XerC and XerD recombinases, and accessory factors ArgR and PepA. It has been suggested that ArgR has a role in the initial pairing of recombination sites and we describe here an attempt to detect this process in vivo. Our approach exploits a previous observation that a cer-like site known as the type II hybrid supports inter-molecular recombination and causes extensive multimerization of plasmids. We report that type-II-mediated multimerization can be repressed by a cer site in cis or in trans and propose that this is due to a physical interaction between the sites. If this hypothesis is correct, suppression of multimer formation provides an assay of site pairing. Our results demonstrate that the putative pairing interaction is independent of the topological relationship of the sites and that both PepA and ArgR are involved. Although most recombination-deficient mutant derivatives of ArgR are unable to pair recombination sites, we have found two (ArgR110 and ArgR115*) which retain pairing activity. The validity of the pairing hypothesis is discussed in the light of alternative explanations for our data.
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Tactic response of zoospores of the fungus Phytophthora palmivora to solutions of different pH in relation to plant infection
More LessSUMMARYZoospores of the plant pathogen Phytophthora palmivora use a number of tactic responses to target specific infectible regions of host roots. Although the dominant one is believed to be chemotaxis, it has been shown that zoospores of oomycetes may also use the exogenous proton/electrical currents generated by plant roots for guidance. Since these proton currents also generate significant pH gradients in the rhizosphere, the tactic response of zoospores to changes in pH was examined. Using ‘swim-in’ capillary tests, zoospores of P. palmivora were found to be repelled by solutions of high pH and attracted to solutions of low pH, relative to a control at neutrality. This in vitro tactic response was generally consistent with the measured pH at sites of zoospore accumulation around intact and wounded roots. However, the endogenous pH gradient around host roots could be abolished with buffers and this treatment did not affect the extent or pattern of zoospore accumulation. Therefore, detection of root-generated pH gradients is unlikely to have a major role in the homing response of zoospores towards plant roots.
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Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis
More LessSUMMARYMycobacterium avium complex strains and Mycobacterium paratuberculosis are closely related intracellular pathogens affecting humans and animals. M. avium complex infections are a leading cause of morbidity and mortality in AIDS patients, and M. paratuberculosis is the agent of Johne's disease in ruminants. Genetic manipulation of these micro-organisms would facilitate the understanding of their pathogenesis, the construction of attenuated vaccine strains and the development of new drugs and treatment methods. This paper describes the replication of mycobacterial shuttle phasmids and plasmids, and the expression of the firefly luciferase reporter gene in M. avium complex and M. paratuberculosis. The mycobacteriophage TM4 propagated on M. smegmatis or M. paratuberculosis plaqued at the same efficiency on these two mycobacterial hosts. Screening of M. avium complex and M. paratuberculosis clinical isolates with TM4-derived luciferase reporter phages demonstrated that the majority of these isolates were susceptible to TM4. Conditions for introduction of DNA were determined by transfection of M. paratuberculosis with TM4 DNA and applied to isolate kanamycin-resistant transformants of M. avium complex and M. paratuberculosis with Escherichia coli-Mycobacterium shuttle plasmids. Recombinant plasmids were recovered from transformants without apparent loss of DNA sequences. These results provide the basis for the genetic manipulation of these pathogenic mycobacterial species.
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Manipulation of intracellular glycerol and erythritol enhances germination of conidia at low water availability
More LessSUMMARYThe insect pathogens Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus can be effective biocontrol agents when relative humidity (RH) is close to 100%. At reduced water availability, germination of propagules, and therefore host infection, cannot occur. Cultures of B. bassiana, M. anisopliae and P. farinosus were grown under different conditions to obtain conidia with a modified polyol and trehalose content. Conidia with higher intracellular concentrations of glycerol and erythritol germinated both more quickly and at lower water activity (aw) than those from other treatments. In contrast, conidia containing up to 235.7 mg trehalose g-1 germinated significantly (P < 0.05) more slowly than those with an equivalent polyol content but less trehalose, regardless of water availability. Conidia from control treatments did not germinate below 0.951-0.935 aw (. 95.1-93.5% RH). In contrast, conidia containing up to 164.6 mg glycerol plus erythritol g-1 germinated down to 0.887 aw (. 88.7% RH). These conidia germinated below the water availability at which mycelial growth ceases (0.930-0.920 aw). Germ tube extension rates reflected the percentage germination of conidia, so the most rapid germ tube growth occurred after treatments which produced conidia containing the most glycerol and erythritol. This study shows for the first time that manipulating polyol content can extend the range of water availability over which fungal propagules can germinate. Physiological manipulation of conidia may improve biological control of insect pests in the field.
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Effect of phytoplankton cell size on transient-state nitrate and ammonium uptake kinetics
More LessSUMMARYThe uptake of nitrate or ammonium (at a concentration of 10 mol I-1) by marine phytoplankton was studied in relation to cell size. Initial specific nitrate uptake rates by small (35000 m3) and large (130000 m3) cells of the diatom Ditylum brightwellii did not differ significantly. However, the larger cells maintained a high uptake rate for a longer time. Therefore, they accumulated nitrate in a higher biomass-specific pool than the smaller cells. In the dark, this effect was even more pronounced. Two smaller diatom species, Lauderia borealis (7474 m3) and Thalassiosira pseudonana (98 (m3), had lower initial specific nitrate uptake rates and lower intracellular pools. Transient-state ammonium uptake did not result in accumulation of large intracellular pools of ammonium. Theoretically, and on the basis of the presented results, we stress the dualistic functional role of the vacuole. A large vacuole is an effective way for larger algal species to possess a minimum cell nutrient quota/cell surface ratio which is in the range of smaller species. Furthermore, by functioning as a storage reservoir it reduces inhibition of the uptake rate by cytoplasmic accumulated nutrients. The effect of the latter mechanism is that larger algal species are better at nitrate uptake under fluctuating conditions. These results imply that, in nitrogen-controlled marine systems, resource competition under fluctuating nutrient concentrations can only lead to a shift towards larger phytoplankton species if nitrate rather than ammonium is the main nitrogen source. From theoretical considerations it is argued that the maximum growth rate of algae is determined by nutrient assimilation properties rather than by photosynthetic capacity.
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Evidence for a developmentally regulated prespore-specific glutamine synthetase in the cellular slime mould Dictyostelium discoideum
More LessSUMMARYThe enzyme glutamine synthetase (GS) is described for the first time in Dictyostelium discoideum. The appearance of this enzyme is developmentally regulated. The level of activity is low in vegetative cells and increases more than threefold during differentiation. Furthermore this enzyme is shown to be differentially localized in prespore cells, the specific activity being approximately fourfold higher than in prestalk cells. The enzyme has a pH optimum of 7.8 and 8.2 in the γ-glutamyltransferase and γ-glutamylsynthetase assays, respectively, and a temperature optimum of 45°C. Kinetic studies of GS revealed apparent Km values of 5.9 mM, 0.009 mM and 8.6 mM for glutamine, ADP and NH2OH, respectively, in the γ-glutamyltransferase assay, and of 2.2 mM, 0.12 mM and 0.64 mM for glutamate, ATP and NH2OH, respectively, in the γ-glutamylsynthetase assay.
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pilO, a gene required for glycosylation of Pseudomonas aeruginosa 1244 pilin
More LessSUMMARYNucleotide sequencing of a region downstream from the Pseudomonas aeruginosa 1244 pilin structural gene, pilA, revealed an ORF potentially able to code for a protein of M r 50862. This ORF, called pilO, was flanked by a tRNAthr gene, which was followed by a transcriptional termination sequence. The tRNAthr gene and the termination sequence were nearly identical to sequences found immediately adjacent to the pilA gene of several P. aeruginosa strains. A 2200 base mRNA strand, which contained both the pilO and pilA transcripts, was produced from this region, while a 650 base transcript containing only pilA was present in a 100-fold excess over the longer transcript. Hyperexpression of the pilA gene in a PilO- strain resulted in normal pilus-specific phage sensitivity and twitching motility. The pilin produced by this strain had a lower apparent M r and a more neutral pI compared to that produced by a strain containing a functional pilO gene. This pilin failed to react with a sugar-specific reagent which recognized pilin produced by the strain containing a functional pilO gene.
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The signal transducer encoded by ampG is essential for induction of chromosomal AmpC -lactamase in Escherichia coli by -lactam antibiotics and unspecific inducers
More LessSUMMARYChemical mutagenesis of the AmpC -lactamase-hyperinducible Escherichia coli strain SN0301/pNu305 carrying the cloned ampC and ampR genes from Citrobacter freundii OS60 gave four independent mutants in which -lactamase was no longer inducible, or was inducible only to a low level, by -lactam antibiotics. The genes ampC, ampR, ampD and ampE, which were essential for -lactamase induction, were functional in these mutants. In all four mutants, the sites of mutation were mapped to 9.9 min on the E. coli chromosome. Complementation with wild-type ampG restored inducibility of -lactamase to wild-type levels. The nucleotide sequence of all four mutant ampG alleles (ampG1, ampG3, ampG4 and ampG5) was determined. In three of the mutants, a single base exchange led to an amino acid change from glycine to aspartate at different sites in the deduced amino acid sequence. In the fourth mutant (ampG4), with low-level inducibility, the nucleotide sequence was identical to wild-type ampG. Spontaneous back-mutation of the chromosomal ampG1 mutant resulted in restoration of wild-type inducibility and a return to the wild-type ampG sequence. Unspecific induction by components of the growth medium was also dependent on intact ampG function.
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Volume 135 (1989)
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Volume 134 (1988)
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Volume 133 (1987)
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Volume 132 (1986)
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Volume 131 (1985)
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Volume 130 (1984)
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Volume 129 (1983)
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Volume 128 (1982)
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Volume 127 (1981)
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Volume 126 (1981)
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Volume 125 (1981)
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Volume 124 (1981)
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Volume 123 (1981)
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Volume 122 (1981)
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Volume 121 (1980)
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Volume 120 (1980)
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Volume 119 (1980)
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Volume 118 (1980)
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Volume 117 (1980)
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Volume 116 (1980)
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Volume 115 (1979)
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Volume 114 (1979)
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Volume 113 (1979)
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Volume 112 (1979)
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Volume 111 (1979)
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Volume 110 (1979)
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Volume 109 (1978)
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Volume 108 (1978)
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Volume 107 (1978)
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Volume 106 (1978)
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Volume 105 (1978)
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Volume 104 (1978)
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Volume 103 (1977)
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Volume 102 (1977)
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Volume 101 (1977)
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Volume 100 (1977)
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Volume 99 (1977)
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Volume 98 (1977)
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Volume 97 (1976)
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Volume 96 (1976)
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Volume 95 (1976)
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Volume 94 (1976)
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Volume 93 (1976)
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Volume 92 (1976)
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Volume 91 (1975)
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Volume 90 (1975)
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Volume 89 (1975)
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Volume 88 (1975)
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Volume 87 (1975)
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Volume 86 (1975)
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Volume 85 (1974)
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Volume 84 (1974)
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Volume 83 (1974)
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Volume 82 (1974)
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Volume 81 (1974)
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Volume 80 (1974)
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Volume 79 (1973)
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Volume 78 (1973)
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Volume 77 (1973)
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Volume 76 (1973)
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Volume 75 (1973)
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Volume 74 (1973)
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Volume 73 (1972)
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Volume 72 (1972)
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Volume 71 (1972)
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Volume 70 (1972)
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Volume 69 (1971)
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Volume 68 (1971)
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Volume 67 (1971)
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Volume 66 (1971)
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Volume 65 (1971)
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Volume 64 (1970)
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Volume 63 (1970)
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Volume 62 (1970)
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Volume 61 (1970)
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Volume 60 (1970)
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Volume 59 (1969)
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Volume 58 (1969)
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Volume 57 (1969)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)