- Volume 137, Issue 4, 1991
Volume 137, Issue 4, 1991
- Review Article
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- Biochemistry
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Characterization of Fonsecaea pedrosoi melanin
More LessSummary: The constituents of the melanin complex from mycelial forms of Fonsecaea pedrosoi were partially characterized. The pigment was mainly accumulated on large alkali-extractable, electron-dense cytoplasmic bodies (melanosomes) and, apparently, on the outer layer of the cell wall as external deposits within verrucose outgrowths. Using electron microscopy and Thiéry’s periodate/thio-semicarbazide/silver proteinate staining method, glycogenlike particles were also detected at the periphery of the cells. Melanin constituents comprised aromatic and aliphatic/glycosidic structures with a predominance of the latter. Infrared spectra showed the presence of hydroxyl, carbonyl and carboxyl groups. The aliphatic/glycosidic moiety consisted of fatty acids and polysaccharides with protein, in a ratio protein/polysaccharide 1:15. Rhamnose, mannose, galactose and glucose (in the ratio 1:2:4:3·5) were the constituents of the polysaccharide. Lipid components included even-numbered, saturated and unsaturated fatty acids (in the ratio 2:1) ranging from C16 to C18. Palmitic and oleic acids were the prominent fatty acids. Aspartic and glutamic acids, leucine, glycine and alanine were the major amino acids. Non-pigmented cells of F. pedrosoi were studied for comparison with the pigmented forms: they did not accumulate acid-insoluble precursors of melanin.
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Occurrence and enzymic degradation of glutathionylspermidine in Citrobacter freundii
More LessSummary: Cells of Citrobacter freundii IFO 12681 contained glutathionylspermidine at a level of 2–3 nmol (g wet cells)−1. Three kinds of enzymes (E1, E2 and E3) catalysing the degradation of glutathionylspermidine were found in extracts of the bacterium. E1 was a spermidine dehydrogenase and was induced by spermidine. The enzyme hydrolysed glutathionylspermidine only in the presence of an electron acceptor. E2 and E3 were formed constitutively and required no cofactor for the degradation of glutathionylspermidine. E3 required at least two protein components for activity. The reaction products formed by E1, E2 and E3 differed from each other, but have not yet been identified. No activity generating glutathione and spermidine from glutathionylspermidine was detected in extracts of C. freundii.
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Efficient translation of synthetic and natural mRNAs in an mRNA-dependent cell-free system from the dimorphic fungus Candida albicans
More LessSummary: An mRNA-dependent cell-free translation system has been developed from the human pathogenic fungus Candida albicans using either S30 or S100 lysates prepared from glass-bead-disrupted whole cells. Translation of the synthetic template poly(U) in this system is highly efficient at temperatures up to 37 °C and is ATP-dependent. Studies using a range of elongation-specific inhibitors suggest that the mechanism of translational elongation in C. albicans is similar to that of another yeast, Saccharomyces cerevisiae. A micrococcal-nuclease-treated C. albicans S100 lysate was able to translate exogenously-supplied homologous mRNAs, and a range of heterologous natural mRNAs, using an initiation mechanism that is inhibited by the antibiotic edeine and the 5′ cap analogue 7-methylguanosine 5′-monophosphate (m7GMP). As with cell-free lysates prepared from S. cerevisiae, the C. albicans lysate is unable to initiate translation upon natural mRNAs at temperatures above 20 °C.
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Fatty acid oxidation and the β-oxidation complex in Mycobacterium leprae and two axenically cultivable mycobacteria that are pathogens
More LessSummary: Intact, non-growing Mycobacterium leprae, M. avium and M. microti oxidized a wide range of 1-14C-labelled fatty acids (C8 to C24) to 14CO2. Laurate (C12) was oxidized most rapidly, and its oxidation by M. leprae was inhibited by the antileprosy agents Dapsone, clofazamine and rifampicin. Key enzymes of β-oxidation were detected in extracts from all three mycobacteria. All these activities (both in intact mycobacteria and the enzymes) were stimulated in M. avium grown in Dubos medium plus palmitate but activities in M. microti or M. avium grown either in Dubos medium with added liposomes or triolein, or in vivo were similar to those detected in the same strain grown in Dubos medium alone. M. avium could be grown in medium in which 95% of its fatty acyl elongase activity is acetyl-CoA dependent. In this medium growing M. avium organisms oxidized [1-14C]palmitate to 14CO2 but simultaneously elongated palmitate to C24 acids and even longer. Acetyl-CoA-dependent elongase activity is similar but clearly not identical to reversed β-oxidation, but the exact point(s) of difference have not yet been identified.
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A conditional-lethal mutant of Bacillus subtilis 168 with a thermosensitive glycerol-3-phosphate cytidylyltransferase, an enzyme specific for the synthesis of the major wall teichoic acid
More LessSummary: A biochemical analysis was undertaken of thermosensitive mutants of Bacillus subtilis 168 harbouring mutations in several tag genes, involved in the synthesis of the major wall teichoic acid, poly(glycerol phosphate), poly(groP). Incorporation of a pulse of [2-3H]glycerol into whole cells, following shift to the restrictive growth temperature, was used to assess synthesis of this polymer and to seek evidence of accumulation of a specific precursor. The rate of incorporation into poly(groP) was strongly decreased in all mutants; glycerol uptake was diminished by 80% or more for a strain harbouring mutation tagB1 (formerly tag-1) and one bearing tagD11 (formerly tag-11). The pool of CDP-glycerol (CDP-gro), a specific precursor of poly(groP), was increased, relative to the wild-type, for all mutations except tagD11, where the pool of CDP-gro was reduced. Cytoplasmic extracts, assayed at the permissive temperature for glycerol-3-phosphate cytidylyltransferase (gro-PCT), the enzyme synthesizing CDP-gro, revealed wild-type activities for all mutations except tagD11. Gro-PCT activity in the latter strain was 100-fold lower and, unlike that in all other mutant strains, highly thermolabile. This thermosensitivity suggests that tagD encodes gro-PCT. The identification, in a gene encoding a poly(groP)-specific enzyme, of a mutation conferring a thermosensitive growth phenotype renders explicit the conclusion that synthesis of this teichoic acid is essential for the growth of B. subtilis.
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Subsite mapping of Hormoconis resinae glucoamylases and their inhibition by gluconolactone
More LessSummary: The subsite maps of two purified glucoamylases (P and S) from the fungus Hormoconis resinae were determined from kinetic data using sets of linear malto-and isomaltooligosaccharides as substrates. Glucoamylase P, which has an unusually high debranching (1,6-glucosidic) activity, showed a subsite map different from all known subsite maps of glucoamylases. The free energy of binding of maltooligosaccharides was negative at subsite 1, whereas all the others show a positive or zero energy at subsite 1. Inhibition of both glucoamylases P and S acting on either malto- or isomaltohexaose by gluconolactone [d-glucono-(1,5)-lactone] was investigated. Gluconolactone decreased the values of the maximum velocity, V, suggesting it can bind to subsite 1. The size of inhibition constants, identified as dissociation constants of gluconolactone from free enzyme, depended on whether the substrate was maltohexaose or isomaltohexaose. This suggests that gluconolactone has at least two binding sites, and that there are different subsites 1 (or 2) for 1,4- and 1,6-linked substrates. From previously reported results with other glucoamylases, an “induced fit” model was constructed for glucoamylases hydrolysing oligosaccharides.
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- Development And Structure
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Differential gene expression and evidence of selective translation during anaerobic germination of Mucor racemosus sporangiospores
More LessSummary: Anaerobically-germinated sporangiospores of Mucor racemosus develop into yeasts, whereas aerobicallygerminated sporangiospores become hyphae. Anaerobic germination was found to have the following traits in common with the previously characterized aerobic system of development: (i) immediate and vigorous protein synthesis upon exposure of the spores to liquid medium; (ii) a complete absence of RNA synthesis for the first 20 min of germination; and (iii) a mobilization of ribosomal subunits into active polyribosomes. The proteins synthesized in both systems during this 20 min interval must be specified by the pre-formed stable mRNA known to be stored in the dormant spore. The population of proteins manufactured early in anaerobic germination differed considerably from the set of proteins synthesized during the equivalent interval in air, suggesting that some unknown mechanism of selective translation must operate. A few dozen of the more prominent proteins could be categorized according to their patterns of synthesis during germination. This should allow future work to focus upon those genes and their products most closely linked to development. The most promising candidates include several proteins that are most conspicuous in the mature yeast and are among those proteins selectively translated in large amounts from stored mRNA templates during anaerobic germination.
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- Genetics And Molecular Biology
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Biochemical and genetic analysis of Streptococcus mutans α-galactosidase
More LessThe aga gene coding for α-galactosidase in Streptococcus mutans was detected in a recombinant gene library constructed in phage λ. The gene was subcloned into plasmid vectors and shown to specify a novel protein of M r 80000. Characterization of α-galactosidase from S. mutans and from recombinant Escherichia coli expressing aga indicated that the enzyme functions as a tetramer. The amino acid composition of the α-galactosidase, deduced from nucleotide sequencing of aga, gave a predicted M r of 82022 and revealed regions of homology to α-galactosidases encoded by the E. coli Raf plasmids and by Bacillus stearothermophilus. Inactivation of the aga gene in S. mutans resulted in loss of all α-galactosidase activity and abolished the ability to ferment melibiose; α-glucosidase activity was also lost, due to an indirect effect on the dexB gene.
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Biochemical and genetic studies on the function of, and relationship between, the PGI1- and CDC30-encoded phosphoglucose isomerases in Saccharomyces cerevisiae
More LessIsoelectric focusing was used to compare the complement of phosphoglucose isomerase isoenzymes in a wild-type strain of Saccharomyces cerevisiae and in a strain with a deletion in the PGI1 structural gene. Deletion of the PGI1 gene did not result in the absence of the high-K m isoenzyme I but the low-K m isoenzyme II was absent. Hence, the isoenzymes must be the products of two genes. If PGI1 were the sole structural gene its deletion would result in the disappearance of both isoenzymes. After a temperature shift-up a cdc30-bearing strain had cell cycle arrested and contained only 8% of the polysaccharide in the wild-type. Phosphoglucose isomerase is required for the synthesis of fructose 6-phosphate (F6-P), a precursor of the cell wall components chitin and mannoprotein (‘mannan’), which are a polysaccharide and contain polysaccharide, respectively. Since the cdc30 mutation confers a temperature-sensitive phosphoglucose isomerase, the likely explanation for cell cycle arrest caused by this mutation is that the defective phosphoglucose isomerase results in a reduction of F6-P and hence an inability to synthesize the mannan and chitin needed for cytokinesis and cell separation. Revertants of a pgi1-1 bearing strain were selected for their ability to grow on glucose at 25 °C and this yielded a number of different phenotypes. Amongst the isolates was a strain which had undergone an intragenic reversion at the pgi1 locus, designated pgi1-1,100. This mutation permits growth and cell division at 25 °C but results in cell cycle arrest at 36 °C. These results are all consistent with the notion that in S. cerevisiae there are two genes (PGI1 and CDC30) which encode the low-K m and high-K m isoenzymes, respectively, of phosphoglucose isomerase and that the two gene products physically interact for activity.
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Expression and secretion of staphylococcal nuclease in yeast: effects of amino-terminal sequences
More LessStaphylococcus aureus nuclease A hybrid genes, encoding proteins OmpA-nuclease, lipo-nuclease and Pin-nuclease, were cloned downstream of the yeast GAL 10 inducible promoter. OmpA-nuclease and lipo-nuclease contain the mature staphylococcal nuclease sequence preceded by the Escherichia coli OmpA and lipoprotein signal sequences, respectively, whereas Pin-nuclease lacks a defined signal sequence at its amino terminus. We found that: (a) the nuclease gene products synthesized in yeast are active, but they do not affect cell growth; (b) OmpA-nuclease and lipo-nuclease are partially processed and constitute approximately 1·0–1·5% of the yeast cell protein; (c) OmpA and lipoprotein signal sequences function similarly in secretion, allowing 35–40% of the processed nuclease to be translocated into the yeast periplasm; and (d) Pin-nuclease, which lacks hydrophobic sequences at its amino-terminus, is accumulated at a level tenfold lower than the hybrid proteins that do contain signal sequences. Nevertheless, 50 % of the enzyme activity of Pin-nuclease in yeast is localized in the periplasmic space.
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Isolation and characterization of sporulation lacZ fusion mutants of Bacillus megaterium
More LessSummary: A derivative of Bacillus megaterium QM B1551 cured of all seven resident plasmids was mutated to Lac–2. Transposon Tn917 carrying lacZ and cat was then used to isolate four spo: :lacZ-cat fusion mutants by screening for colonies expressing the fusion in stationary phase. The sporulation frequencies of the mutants ranged from 10–7 to 10–2. Macrolide, lincosamide and streptogramin B resistance (MLSr) of the mutants cotransduced 100% with the sporulation defect and all four mutations mapped near trp by transduction in the order: trp–hisH–spo. All four mutants isolated were defective early in sporulation since β-galactosidase could be detected between zero and 2 h after the end of exponential growth. Electron microscopy of two of the mutants expressing the enzyme at t1–t2 revealed a defect prior to or just at the beginning of septum formation in one, and after completion of septum formation in the other. Little or no synthesis of dipicolinic acid, glucose dehydrogenase or alkaline phosphatase was detected in the mutants, but each was neutral protease positive. These results show that the mutations were in at least two early genes expressed before glucose dehydrogenase production. This study represents the first genetic characterization of sporulation mutants in B. megaterium and also demonstrates that gene fusion technology can be used in this species.
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Characterization of transposon insertion mutants of Methylobacterium extorquens AM1 (Methylobacterium strain AM1) which are defective in methanol oxidation
More LessSummaryThree previously reported Tn5 mutants of Methylobacterium extorquens AM1, Cou-1, Cou-3 and Cou-6, which are able to grow on methylamine but not methanol, were characterized by biochemical analyses and complementation tests using two genomic libraries of M. extorquens AM1. We have designated the genes defective in these mutants as cou-1, cou-3 and cou-6 and mapped the site of Tn5 insertion in each. Biochemical results showed that two of these methanol oxidation (Mox) mutants, Cou-1 and Cou-3, are phenotypically similar to the previously identified MoxE class of mutant while Cou-6 resembled the MoxD class. Complementation tests and mapping the site of the Tn5 insertions indicated that cou-1 is another Mox gene linked to moxE and that cou-6 is linked to moxD. The Tn5 insertion in cou-3 mapped within the moxJ gene and therefore is the first detected mutation of the moxJ gene which was identified from expression studies. A MoxE mutant of M. extorquens AM1 was complemented by two different cosmid clones; one carried the moxE gene and the other contained two mox gene clusters, moxFJGI and moxAKLB. Hybridization experiments indicated that the moxE gene was not present on this latter clone, and it must therefore encode a gene capable of suppressing a MoxE mutation.
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Genes concerned with synthesis of poly(glycerol phosphate), the essential teichoic acid in Bacillus subtilis strain 168, are organized in two divergent transcription units
More LessSummary: Insertional mutagenesis has revealed that a 22 kbp segment from the hisA region of the Bacillus subtilis 168 chromosome (310° on the genetic map) contains at least six independent transcription units, all apparently devoted to production of cell envelope components. Genes concerned with synthesis of poly(glycerol phosphate), poly(groP), an essential cell wall polymer in B. subtilis 168, are organized in two divergently transcribed operons denoted tagABC and tagDEF. Nucleotide sequence analysis indicates that three of these six genes encode extremely basic polypeptides. The deduced products of the tagABC operon may be involved in poly(groP) assembly and export, whereas those of the tagDEF operon, which are very hydrophilic, are more likely to be implicated in poly(groP) precursor biosynthesis. The first gene of the tagDEF operon encodes glycerol-3-phosphate cytidylyltransferase ( Pooley et al., 1991 , Journal of General Microbiology 137, 921–928) and its deduced product has significant homology with cholinephosphate cytidylyltransferase from yeast. There is also substantial homology between the deduced products of tagB in the tagABC operon and tagF in the tagDEF operon.
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Genetic and biochemical characterization of Bacillus subtilis 168 mutants specifically blocked in the synthesis of the teichoic acid poly(3-O-β-d-glucopyranosyl-N-acetylgalactosamine 1-phosphate): gneA, a new locus, is associated with UDP-N-acetylglucosamine 4-epimerase activity
More LessSummary: The resistance spectrum to bacteriophase ø3T of different Bacillus subtilis 168/W23 strains hybrid for wall teichoic acids suggested that poly(3-O-β-d-glucopyranosyl-N-acetylgalactosamine 1-phosphate), a so-called minor teichoic acid of strain 168, forms part of the receptor for this phage, and a serologically related group of phages. A representative sample of 25 mutants specifically resistant to ø3T, obtained from a mutagenized culture by direct selection, were all found to have a greatly reduced galactosamine content. Relevant mutations in these strains were shown by PBS1 transduction and transformation to belong to two linkage groups; a minority, associated with an atypical colony morphology, were localized between sacA and purA, wherease the majority mapped between gtaB and tagB1 (formerly tag-1), a region containing all known genes involved in the synthesis of the major wall teichoic acid, poly(glycerol phosphate). The fomer mutations mapped in a new locus, gneA, characterized by a deficiency in UDP-N-acetylglucosamine 4-epimerase, while the latter ones, as well as the previously identified pha-3 ( Estrela et al., 1986 , Journal of General Microbiology 132, 411–415), map in a lucus named gga. They are likely to affect membrane-bound enzymes involved in the synthesis of the galactosamine-containing teichoic acid. A possible biological role of this polymer is discussed.
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Genes involved in meso-diaminopimelate synthesis in Bacillus subtilis: identification of the gene encoding aspartokinase I
More LessSummary: Thermosensitive mutants of Bacillus subtilis deficient in peptidoglycan synthesis were screened for mutations in the meso-diaminopimelate (ld-A2pm) metabolic pathway. Mutations in two out of five relevant linkage groups, IssB and IssD, were shown to induce, at the restrictive temperature, a deficiency in ld-A2pm synthesis and accumulation of UDP-MurNAc-dipeptide. Group IssB is heterogeneous; it encompasses mutations that confer deficiency in the deacylation of N-acetyl-ll-A2pm and accumulation of this precursor. Accordingly, these mutations are assigned to the previously identified locus dapE. Mutations in linkage group IssD entail a thermosensitive aspartokinase I. Therefore, they are most likely to affect the structural gene of this enzyme, which we propose to designate dapG. Mutation pyc-1476, previously reported to affect the pyruvate carboxylase, was shown to confer a deficiency in aspartokinase I, not in the carboxylase, and to belong to the dapG locus. dapG is closely linked to spoVF, the putative gene of dipicolinate synthase. In conclusion, mutations affecting only two out of eight steps known to be involved in ld-A2pm synthesis were uncovered in a large collection of thermosensitive mutants obtained by indirect selection. We propose that this surprisingly restricted distribution of the thermosensitive dap mutations isolated so far is due to the existence, in each step of the pathway, of isoenzymes encoded by separate genes. The biological role of different aspartokinases was investigated with mutants deficient in dapE and dapG genes. Growth characteristics of these mutants in the presence of various combinations of aspartate family amino acids allow a reassessment of a metabolic channel hypothesis, i.e. the proposed existence of multienzyme complexes, each specific for a given end product.
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Ammonium transport in Escherichia coli: localization and nucleotide sequence of the amtA gene
More LessSummary: Escherichia coli expresses a concentrative ammonium (methylammonium) transport system which is strongly repressed under conditions of nitrogen excess. We have previously reported the cloning of a structural gene (amtA) for this transporter by complementation. In this study, a 3·4 kb HindIII-BamHI fragment containing amtA was cloned into the pBluescript KS(+) vector, and unidirectional nested deletions from each end of this 3·4 kb fragment were generated by exonuclease III digestion. The deletions were analysed by complementation of the structural gene mutation produced by Tn10 insertion. This allowed amtA to be localized within a 1·4 kb region which spans the site of the mutation. By application of the Sanger dideoxy method, we sequenced the region containing amtA. The gene contains an open reading frame which encodes a protein with a predicted molecular mass of 27 kDa. The open reading frame is preceded by a putative Shine?Dalgarno sequence and followed by an inverted repeat which might function as a simple transcription terminator. Hydropathic analysis of the inferred amino acid sequence of the gene product predicts that amtA encodes a cytoplasmic component of the ammonium transport system.
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Cloning, sequencing, and mutational analysis of the Bradyrhizobium japonicum fumC-like gene: evidence for the existence of two different fumarases
More LessSummary: The Bradyrhizobium japonicum fumarase gene (fumC-like) was cloned and sequenced, and a fumC deletion mutant was constructed. This mutant had a Nod+ Fix+ phenotype in symbiosis with the host plant, soybean, and growth in minimal medium with fumarate as sole carbon source was also not affected. The cloned B. japonicum fumC gene fully complemented an Escherichia coli Fum- mutant, strain JH400, for growth in minimal medium with fumarate. The predicted amino acid sequence of the FumC protein showed strong similarity to the E. coli FumC protein, Bacillus subtilis CitG protein, Saccharomyces cerevisiae Fum1 protein, and the mammalian fumarases. The B. japonicum FumC protein accounted for about 40% of the total fumarase activity in aerobically grown cells. The remaining 60% was ascribed to a temperature-labile fumarase. These data suggest that B. japonicum possesses two different fumarase isoenzymes, one of which is encoded by fumC. Besides E. coli, which has three fumarases, B. japonicum is thus the second bacterium for which there is genetic evidence for the existence of more than one fumarase.
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- Pathogenicity And Medical Microbiology
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Effect of iron deprivation on surface composition and virulence determinants of Candida albicans
More LessSummary: Six strains of Candida albicans were grown in defined medium which had been deferrated by ion-exchange chromatography and then supplemented with FeCl3 to give iron concentrations ranging from 0·026 μM to 0·8 μm. Growth in 0-026 μM-iron (measured as increase in biomass) was reduced by 26–59% as compared with that in excess (0·8 μM)iron. With five of the strains, adhesion to buccal epithelial cells was maximal after growth in 0·2–0-4μM-iron, but strain GDH 2023 adhered best when grown in 0·026 μm-iron. Differences in yeast cell-wall composition were revealed by Zymolyase treatment of whole cells and by 125I-labelling of surface proteins. SDS-PAGE of iodinated proteins, followed by autoradiography, showed quantitative but no qualitative differences in protein profiles of iron-deficien and iron-replete organisms. The ability of all strains to form germ tubes in serum was near-maximal after growth in 0·2–0·4 μm-iron but was inhibited by up to 93% following growth in lower results indicate that expression of important virulence attributes by C. albicans is highly iron and that expression in vivo may therefore be significantly different from that observed under conventional laboratory conditions.
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Molecular cloning and characterization of form I antigen genes of Shigella sonnei
More LessSummary: Sau3AI-generated DNA fragments of the Shigella sonnei large plasmid encoding the form I antigen were cloned into Escherichia coli with cosmid vector pHSG262. One resulting plasmid, designated pJK1137, was studied further. Restriction endonuclease mapping and analysis of transposon Tn3 insertion mutants demonstrated that the form I antigen genes were located within a region of about 12·6 kb consisting of the two contiguous HindIII fragments of 1·26 kb and 12·4 kb. The results of complementation studies between Tn3 insertion mutants of pJK1137 and recombinant plasmids carrying different parts of the form I antigen genes indicated that the 12·6 kb DNA sequence contained at least four gene clusters, regions A, B, C and D. Analysis of radioactively labelled proteins in minicells demonstrated that the DNA sequence of about 12·6 kb coded for at least four specific proteins of 42, 23, 48 and 39 kDa. The former two were coded by region A, the latter two by region D.
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A localization index for distinction between extracellular and intracellular antigens of Mycobacterium tuberculosis
More LessA series of mycobacterial antigens were quantified by immunoelectrophoresis, enzyme-linked immunosorbent assays, or SDS-PAGE with immunoblotting using antisera against purified mycobacterial antigens. The antigens showed a characteristic distribution profile. Some had a marked quantitative dominance in the culture fluid while others had a marked dominance in sonicates of whole washed bacilli. The majority of the antigens tested could thus be located and grouped as either secreted or cytoplasmic in terms of a localization index (LI) which is described. A 5-week-old Mycobacterium tuberculosis culture fluid preparation with a low degree of lysis was valuable in the delineation of localization indexes. The various secreted antigens showed a great span in LI values, from 5 to 1000. This variation may express different degrees of secretion efficiency or differences in tendency to adhere to the bacterial surface. The identification of proteins as extracellular or cytosolic according to their LI values was in agreement for cultures of M. tuberculosis with a low degree of lysis and cultures of M. bovis BCG and M. bovis AN-5 with significant lysis of the bacterial cells.
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Characterization and comparative bactericidal activity of monoclonal antibodies to Bordetella pertussis lipo-oligosaccharide A
More LessSummary: Spleen cells from mice immunized with a Bordetella pertussis N-lauroyl sarcosine membrane extract (SME) were used to generate hybridoma cells lines producing monoclonal antibodies (mAbs). Seven mAbs were shown to be specific to B. pertussis lipo-oligosaccharide (LOS) by immunoblotting of SME or purified LOS following SDS-PAGE. All mAbs reacted with the B. pertussis Tohama I strain of the LOS AB phenotype, and did not react with the atypical variant strain 134 of the LOS B phenotype. The immune reactivity of the mAbs was retained after treatment of SME with proteinase K and was lost after sodium periodate treatment. No cross-reactivity was observed with the mAbs when tested against B. parapertussis and other Gram-negative bacteria. However, all mAbs reacted with B. bronchiseptica. Binding assays with live B. pertussis cells demonstrated that mAbs strongly reacted with cell surface exposed antigenic determinants. High bacterial cell lytic capability was observed for five of these mAbs. Concentrations between 0·22 and 2·2 μg mAb ml–1 (0·1 and 1 μg per 450 μl assay) purified by protein A were required to kill at least 50% of the bacteria. Competition immunoassays with biotinylated antibodies showed that the bacteriolytic and non-bacteriolytic mAbs were directed to different epitopes of the B. pertussis LOS A.
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Cloning and sequence analysis of a plasmid-encoded chloramphenicol acetyltransferase gene from Staphylococcus intermedius
More LessSummary: The chloramphenicol acetyltransferase gene (cat) of a 3·9 kb chloramphenicol resistance (CmR) plasmid from Staphylococcus intermedius, designated pSCS1, was cloned into an Escherichia coli plasmid vector. Sequence analysis revealed a high degree of base similarity with the cat gene of the S. aureus CmR plasmid pC221 but there were several differences in the regulatory region. A lesser degree of similarity was observed between the cat gene of the S. intermedius plasmid and the cat gene of the S. aureus plasmid pC194.
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- Physiology And Growth
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The permittistat: a novel type of turbidostat
More LessBaker’s yeast was grown in a novel type of turbidostat in which the steady-state biomass level was controlled not by the optical turbidity but by the dielectric permittivity of the suspension at appropriate radio frequencies. Dry weight, fresh weight, the optical density at 600 nm, percentage viability (from methylene blue staining), bud count and ethanol concentration were measured off-line and the cell size distribution was recorded using flow cytometry. Any changes in the physiological properties of the yeast had a negligible effect on the ratio between the permittivity set (and measured) and the steady-state dry weight, fresh weight or optical density of the cultures. The permittistat was found to provide an extremely convenient means for carrying out turbidostatic culture.
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The role of trehalose in the osmoadaptation of Escherichia coli NCIB 9484: interaction of trehalose, K+ and glutamate during osmoadaptation in continuous culture
More LessNatural abundance 13C nuclear magnetic resonance spectroscopy identified the disaccharide trehalose as the major organic osmolyte synthesized by Escherichia coli grown in continuous culture under nitrogen limitation in the presence of 0·5 m-NaCl. Trehalose accumulation was dependent on both the growth phase of the culture and the osmolality of the growth medium, but independent of the solute used to increase the osmolality as long as the solute was non-penetrant. The penetrant solute glycerol did not induce trehalose synthesis indicating that the loss of cell turgor rather than increasing medium osmolality per se was the mechanism stimulating trehalose synthesis. Under conditions of either carbon or nitrogen limitation osmoadaptation was distinctly biphasic. The initial response consisted of a rapid (within 30 min) accumulation of K+ and a concurrent synthesis of the amino acid glutamate; trehalose synthesis occurred during the second slower phase of osmoadaption. Chloramphenicol severely inhibited trehalose accumulation indicating that the enzyme(s) involved in trehalose synthesis were inducible.
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Lignocellulose biodegradation by Agaricus bisporus during solid substrate fermentation
More LessAgaricus bisporus was cultivated axenically on wheat straw compost. Analysis of this culture medium during growth and fruiting showed that the lignin fraction of straw was degraded preferentially during the vegetative growth phase, whereas cellulose was degraded after the emergence of the fruit bodies. A novel technique was developed, whereby natural or synthetic radiolabelled lignin was mixed intimately with axenic compost and the rate of mineralization to CO2 throughout the life-cycle of A. bisporus was monitored continuously without culture disturbance. Mineralization rates were maximal during the vegetative growth phase and the onset of fruiting brought a decrease of this activity. A mutant strain of A. bisporus, which was unable to develop fruiting bodies, was shown to mineralize radiolabelled lignin continuously.
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Mutants of Bacillus stearothermophilus defective in the uptake and metabolism of acetate
More LessSummary: Mutants unable to utilize acetate as sole source of carbon and energy were isolated from ethyl-methanesulphonate-mutagenized cultures of a prototrophic Bacillus stearothermophilus strain. Three groups of mutants had identifiable defects. Thirteen of the mutants lacked one or both enzymes of the inducible anaplerotic glyoxylate shunt, and three mutants were auxotrophic for isoleucine and valine when grown on acetate, in a manner analogous to acetohydroxyacid synthase I mutants of enteric bacteria. One mutant was defective specifically in acetate uptake, providing direct evidence for an acetate transport system in B. stearothermophilus. The acetate uptake system was uncoupler-sensitive and had a relatively high K m (in the range 300–350 μm). Escherichia coli was also shown to possess a saturable acetate uptake system, but its K m was much lower (15 μm).
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Temperature shifts and sporulation of Bacillus megaterium
More LessSummary: The permissive temperature for sporulation of Bacillus megaterium 27 (up to 42 °C) was found to be 4–5 °C lower than that for growth. The non-permissive temperature suppressed the initial phases of sporulation characterized by the synthesis of an extracellular proteinase but the cells retained the ability to sporulate for several hours. Neither growth at supraoptimal temperatures nor heat shock applied at the end of the growth phase increased the permissive sporulation temperature. The organism synthesized at least ten heat-shock proteins, the dominant one being HSP 69. These proteins were also found in cells after 3 h of incubation at 43·5 °C but their presence did not ensure the ability to sporulate at this temperature. The rise of temperature provoked an imbalance between synthesis and degradation of cellular proteins, whose role in suppression of sporulation is discussed.
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Changes in translatable mRNA species associated with nutrient deprivation and protease synthesis in Metarhizium anisopliae
More LessSummary: We have studied the regulation of the extracellular chymoelastase protease (PrI) of the fungal entomopathogen Metarhizium anisopliae. This enzyme is involved in the penetration of insect cuticle by Metarhizium and other entomopathogenic fungi. Changes in mRNA sequences during starvation-induced synthesis of chymoelastase were investigated by comparing poly(A+)RNAs and their complementary DNA in rapidly growing or nutrient-deprived cultures of Metarhizium. Hybrid-selected translation revealed that three novel polypeptides (41, 40·2 and 29·8 kDa) were produced rapidly (<1 h) during nutrient deprivation; the most intensely translated species (41 kDa) was identified as the primary translation product of Pr1 by examination with anti-Pr1-IgG. Measurement of starvation-specific RNA levels by dot-blot hybridization with [32P]cDNA showed that transcripts were not present in spores but were produced when the formation against a hard surface of infection structures was induced by depletion of endogenous nutrient reserves. Starvation-specific mRNA species failed to appear when new RNA synthesis was blocked with actinomycin D, indicating that control is at the level of transcription. Immunoblot analysis with anti-Pr1-IgG showed that Pr1 protein increased in concert with the appearance of Pr1 transcripts and active enzyme. Two very short lived precursors of Pr1, the primary translation product (41 kDa) and an intermediate (40·8 kDa) in addition to mature Pr1 (30 kDa) were detected by immunoprecipitation of [35S]methionine-labelled cell extracts with anti-Pr1-IgG. Pulse-labelling experiments demonstrated that a time of about 7 min was required for [35S]methionine to be processed into extracellular Pr1. These results suggest that regulation of Pr1 gene expression occurs during starvation which, coupled with fast processing, allows very rapid secretion of Pr1. Isolates of four other entomopathogenic fungi (Verticillium lecanii, Beauveria bassiana, Tolypocladium niveum and Paecilomyces farinosus) produced Pr1-type enzymes during nutrient deprivation, suggesting our results may have widespread application in understanding entomopathogenicity.
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The effect of temperature shifts on protein synthesis by the psychrophilic bacterium Vibrio sp. strain ANT-300
More LessSummary: In the psychrophilic bacterium Vibrio sp. strain ANT-300, the temperature-related characteristics of protein synthesis in cells grown at 0 °C differed from those of cells grown at 13 °C. Cells grown at 0 °C and 13 °C transported amino acids at the same rates, dependent on the temperature at which rates were measured. The rates of protein synthesis in extracts of cells grown at 0 °C and at 13 °C differed, as a result of the changes in the properties of the soluble fraction involved in protein synthesis. Concurrently, levels of more than 24 polypeptides in the soluble fraction changed considerably. These results suggest that the difference in temperature dependence of protein synthesis in cells grown at various temperatures may be brought about by specific changes in the levels of a small number of polypeptides (less than 15% of the total number of proteins detected by silver-staining) in response to a change in temperature.
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Bacteriolytic effect of teicoplanin
More LessSummary: The glycopeptide antibiotic teicoplanin belongs to the same group as vancomycin and ristocetin and is a valuable tool for studying the autolytic system of sensitive Gram-positive bacteria. Teicoplanin, at a concentration of 1 μg ml–1, caused rapid lysis of exponential phase cells of Streptococcus faecalis. Bacillus spp. were most sensitive to the antibiotic; effective lysis occurred at 0·1 μg teicoplanin ml–1. The bacteriolytic effect depended on the antibiotic concentration, the growth phase and growth rate of the target organism. Antibiotic added to overnight cultures did not cause lysis. Mg2+ (50 mm) was unable to prevent lysis. Mutants with decreased autolytic activity were more resistant to teicoplanin and lysed more slowly than the wild-type. Growth of bacteria in slightly acidic medium protected the cells against the lytic effect of teicoplanin typically observed at pH 7 or 8. This pH-dependent antibiotic tolerance was demonstrated with both bacilli and streptococci. Bacterial lysis was prevented by the presence of Ac-l-Lys(Ac)-d-Ala-d-Ala and normal growth was observed when this peptide was added simultaneously with teicoplanin. Bacteria pretreated with teicoplanin, washed and transferred to fresh medium or buffers behaved as if the antibiotic was still present; in neutral or slightly alkaline conditions strong lysis occurred, whereas in acidic buffer only bacteriostasis was observed. In contrast to vancomycin, teicoplanin induced some lysis of bacteria in hypertonic media, presumably by affecting the integrity of the cell membrane.
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Exogenous cAMP and cGMP modulate branching in Fusarium graminearum
More LessSummary: A study was made of the effects of adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP) and choline on the morphology and growth of a wild-type strain (A 3/5) and a highly branched, “colonial” mutant strain (C106) of Fusarium graminearum. Addition of up to 50 mm-cAMP or cGMP to the medium had no effect on the specific growth rate of strain A 3/5. For strain A 3/5, but not for strain C106, exogenous cAMP caused significant decreases in both mean hyphal extension rate (E) and hyphal growth unit length (G), i.e. cAMP caused mycelia of strain A 3/5 to branch profusely. By contrast, for both strains, cGMP caused significant increases in both E and G, i.e. exogenous cGMP caused mycelia to branch more sparsely. The effects of exogenous cGMP and choline in increasing E and G were synergistic, but the effects of cGMP and choline counteracted the effect of cAMP. The mutant phenotype of strain C106 was not correlated with altered levels of endogenous cAMP or cGMP.
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Changes in the activities of key enzymes of glycolysis during the cell cycle in yeast: a rectification
More LessSummary: Activities of glycolytic enzymes were determined in elutriation fractionated cultures of Saccharomyces cerevisiae grown on different carbon sources. Almost pure fractions of single cells at the G1 stage of cell division were obtained for some of the growth conditions tested, whereas other stages were enriched in particular fractions. Specific activities of glucose-6-phosphate dehydrogenase and alcohol dehydrogenase were found to be constant during the cell cycle, as reported by van Doorn et al. (1988a) , Journal of Bacteriology 170, 4808–4815, and (1988b), Journal of General Microbiology 134, 785–790. In contrast to the earlier reports, the activities of hexokinase, phosphofructokinase, pyruvate kinase and trehalase were also constant in different stages of the cell cycle. For hexokinase and phosphofructokinase it was shown that the apparent specific activity in a cell-free extract strongly diminished when extracts contained less than 0·5-1 mg protein ml−1. In the experiments of van Doorn et al. (1988a) the protein content of the outer fractions was up to 20 times lower than that of the central fractions, suggesting an alternative explanation for the observed changes in enzyme activities during the cell cycle. Therefore, we want to rectify the observations presented by van Doorn et al. (1988a) , and conclude that the activities of the glycolytic enzymes do not vary greatly during the cell cycle of S. cerevisiae.
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