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Volume 149,
Issue 9,
2003
Volume 149, Issue 9, 2003
- Microbiology Comment
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- Cell And Developmental Biology
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Calcium gradient dependence of Neurospora crassa hyphal growth
More LessA tip-high cytoplasmic calcium gradient has been identified as a requirement for hyphal growth in the fungus Neurospora crassa. The Ca2+ gradient is less steep compared to wall vesicle, wall incorporation and vesicular Ca2+ gradients, but this can be explained by Ca2+ diffusion. Analysis of the relation between the rate of hyphal growth and the spatial distribution of tip-localized calcium indicates that hyphal growth rates depend upon the tip-localized calcium concentration. It is not the steepness of the calcium gradient, but tip-localized calcium and the difference in tip-localized calcium versus subapical calcium concentration which correlate closely with hyphal growth rate. A minimal concentration difference between the apex and subapical region of 30 nM is required for growth to occur. The calcium concentration dependence of growth may relate directly to biochemical functions of calcium in hyphal extension, such as vesicle fusion and enzyme activation during cellular expansion. Initiation of tip growth may rely upon random Ca2+ motions causing localized regions of elevated calcium. Continued hyphal expansion may activate a stretch-activated phospholipase C which would increase tip-localized inositol 1,4,5-trisphosphate (IP3). Hyphal expansion, induced by mild hypoosmotic treatment, does increase diacylglycerol, the other product of phospholipase C activity. This is consistent with evidence that IP3-activated Ca2+ channels generate and maintain the tip-high calcium gradient.
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A synthetic analysis of the Saccharomyces cerevisiae stress sensor Mid2p, and identification of a Mid2p-interacting protein, Zeo1p, that modulates the PKC1–MPK1 cell integrity pathway
More LessMid2p is a plasma membrane protein that functions in Saccharomyces cerevisiae as a sensor of cell wall stress, activating the PKC1–MPK1 cell integrity pathway via the small GTPase Rho1p during exposure to mating pheromone, calcofluor white, and heat. To examine Mid2p signalling, a global synthetic interaction analysis of a mid2 mutant was performed; this identified 11 interacting genes. These include WSC1 and ROM2, upstream elements in cell integrity pathway signalling, and FKS1 and SMI1, required for 1,3-β-glucan synthesis. These synthetic interactions indicate that the Wsc1p sensor acts through Rom2p to activate the Fks1p glucan synthase in a Mid2p-independent way. To further explore Mid2p signalling a two-hybrid screen was done using the cytoplasmic tail of Mid2p; this identified ZEO1 (YOL109w), encoding a 12 kDa peripheral membrane protein that localizes to the plasma membrane. Disruption of ZEO1 leads to resistance to calcofluor white and to a Mid2p-dependent constitutive phosphorylation of Mpk1p, supporting a role for Zeo1p in the cell integrity pathway. Consistent with this, zeo1-deficient cells suppress the growth defect of mutants in the Rho1p GDP–GTP exchange factor Rom2p, while exacerbating the growth defect of sac7Δ mutants at 37 °C. In contrast, mid2Δ mutants have opposing effects to zeo1Δ mutants, being synthetically lethal with rom2Δ, and suppressing an 18 °C growth defect of sac7Δ, while overexpression of MID2 rescues a rom2Δ 37 °C growth defect. Thus, MID2 and ZEO1 appear to play reciprocal roles in the modulation of the yeast PKC1–MPK1 cell integrity pathway.
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Induction of L-form-like cell shape change of Bacillus subtilis under microculture conditions
A remarkable cell shape change was observed in Bacillus subtilis strain 168 under microculture conditions on CI agar medium (Spizizen's minimal medium supplemented with a trace amount of yeast extract and Casamino acids). Cells cultured under a cover glass changed in form from rod-shaped to spherical, large and irregular shapes that closely resembled L-form cells. The cell shape change was observed only with CI medium, not with Spizizen's minimum medium alone or other rich media. The whole-cell protein profile of cells grown under cover glass and cells grown on CI agar plates differed in several respects. Tandem mass analysis of nine gel bands which differed in protein expression between the two conditions showed that proteins related to nitrate respiration and fermentation were expressed in the shape-changed cells grown under cover glass. The cell shape change of CI cultures was repressed when excess KNO3 was added to the medium. Whole-cell protein analysis of the normal rod-shaped cells grown with 0·1 % KNO3 and the shape-changed cells grown without KNO3 revealed that the expression of the branched-chain α-keto acid dehydrogenase complex (coded by the bfmB gene locus) was elevated in the shape-changed cells. Inactivation of the bfmB locus resulted in the repression of cell shape change, and cells in which bfmB expression was induced by IPTG did show changes in shape. Transmission electron microscopy of ultrathin sections demonstrated that the shape-changed cells had thin walls, and plasmolysis of cells fixed with a solution including 0·1 M sucrose was observed. Clarifying the mechanism of thinning of the cell wall may lead to the development of a new type of cell wall biosynthetic inhibitor.
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Shigella-induced necrosis and apoptosis of U937 cells and J774 macrophages
More LessIt is currently unclear whether Shigella kills its phagocytic host cells by apoptosis or necrosis. This study shows that rapid necrosis ensues in macrophage-like cell lines (U937 cells differentiated by all-trans-retinoic acid and J774 cells) infected with the Shigella flexneri strain YSH6000. The infected cells rapidly lose membrane integrity, a typical feature of necrosis, as indicated by the release of the cytoplasmic lactate dehydrogenase and the exposure of phosphatidylserine (PS) associated with the rapid uptake of propidium iodide (PI). The infected cells exhibit DNA fragmentation without nuclear condensation, and substantial involvement of either caspase-3/-7 or caspase-1 was not detected, which is also contrary to what is normally observed in apoptosis. Cytochalasin D potently inhibited Shigella-induced cell death, indicating that only internalized Shigella can cause necrosis. Osmoprotectants such as polyethylene glycols could suppress cell death, suggesting that insertion of a pore by Shigella into the host cell membrane induces the necrosis. The pore was estimated to be 2·87±0·4 nm in diameter. Shigella was also found to be able to induce apoptosis but only in one of the lines tested and under specific conditions, namely U937 cells differentiated with interferon-γ (U937IFN). Caspase-3/-7 but not caspase-1 activation was observed in these infected cells and the exposure of PS occurred without the uptake of PI. An avirulent Shigella strain, wild-type Shigella killed with gentamicin, and even Escherichia coli strain JM109, could also induce apoptosis in U937IFN cells, and cytochalasin D could not prevent apoptosis. It appears therefore that Shigella-induced apoptosis of U937IFN cells is unrelated to Shigella pathogenicity and does not require bacterial internalization. Thus, Shigella can induce rapid necrosis of macrophage-like cells in a virulence-related manner by forming pores in the host cell membrane while some cells can be killed through apoptosis in a virulence-independent fashion.
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- Biochemistry And Molecular Biology
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The Bacillus subtilis YufLM two-component system regulates the expression of the malate transporters MaeN (YufR) and YflS, and is essential for utilization of malate in minimal medium
More LessThe Gram-positive bacterium Bacillus subtilis has a complete set of enzymes for the tricarboxylic acid (TCA) cycle and can grow aerobically using most of the TCA cycle intermediates (malate, fumarate, succinate and citrate) as a sole carbon source. The B. subtilis genome sequence contains three paralogous two-component regulatory systems, CitST, DctSR and YufLM. CitST and DctSR activate the expression of a transporter of the Mg2+–citrate complex (CitM) and a fumarate and succinate transporter (DctP), respectively. These findings prompted an investigation of whether the YufL sensor and its cognate regulator, YufM, play a role in malate uptake. This paper reports that the YufM regulator shows in vitro binding to the promoter region of two malate transporter genes, maeN and yflS, and is responsible for inducing their expression in vivo. It was also found that inactivation of the yufM or maeN genes resulted in bacteria that could not grow in a minimal salts medium containing malate as a sole carbon source, indicating that the induction of the MaeN transporter by the YufM regulator is essential for the utilization of malate as a carbon source. Inactivation of the yufL gene resulted in the constitutive expression of MaeN. This expression was suppressed by reintroduction of the kinase domain of YufL, indicating that the YufL sensor is required for proper signal detection and signalling specificity. The authors propose that a phosphatase activity of YufL plays an important role in the YufLM two-component regulatory system. The studies reported here have revealed that members of a set of paralogous two-component regulatory systems in B. subtilis, CitST, DctSR and YufLM, are involved in a related function – uptake (and metabolism) of the TCA cycle intermediates – but with distinct substrate specificities.
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The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate
A transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene. The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (k cat/K m 102 and 10 s−1 mM−1, respectively). However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression. Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. ywkA and yufL/yufM could thus be renamed maeA for malic enzyme and malK/malR for malate kinase sensor/malate response regulator, respectively.
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Distinct molecular mechanisms involved in carbon catabolite repression of the arabinose regulon in Bacillus subtilis
More LessThe Bacillus subtilis proteins involved in the utilization of l-arabinose are encoded by the araABDLMNPQ–abfA metabolic operon and by the araE/araR divergent unit. Transcription from the ara operon, araE transport gene and araR regulatory gene is induced by l-arabinose and negatively controlled by AraR. Additionally, expression of both the ara operon and the araE gene is regulated at the transcriptional level by glucose repression. Here, by transcriptional fusion analysis in different mutant backgrounds, it is shown that CcpA most probably complexed with HPr-Ser46-P plays the major role in carbon catabolite repression of the ara regulon by glucose and glycerol. Site-directed mutagenesis and deletion analysis indicate that two catabolite responsive elements (cres) present in the ara operon (cre araA and cre araB) and one cre in the araE gene (cre araE) are implicated in this mechanism. Furthermore, cre araA located between the promoter region of the ara operon and the araA gene, and cre araB placed 2 kb downstream within the araB gene are independently functional and both contribute to glucose repression. In Northern blot analysis, in the presence of glucose, a CcpA-dependent transcript consistent with a message stopping at cre araB was detected, suggesting that transcription ‘roadblocking’ of RNA polymerase elongation is the most likely mechanism operating in this system. Glucose exerts an additional repression of the ara regulon, which requires a functional araR.
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Membrane-bound hydrogenase and sulfur reductase of the hyperthermophilic and acidophilic archaeon Acidianus ambivalens
More LessA sulfur reductase (SR) and a hydrogenase were purified from solubilized membrane fractions of anaerobically grown cells of the sulfur-dependent archaeon Acidianus ambivalens and the corresponding genes were sequenced. The SR reduced elemental sulfur with hydrogen as electron donor [45 U (mg protein)−1] in the presence of hydrogenase and either 2,3-dimethylnaphthoquinone (DMN) or cytochrome c in the enzyme assay. The SR could not be separated from the hydrogenase during purification without loss of activity, whereas the hydrogenase could be separated from the SR. The specific activity of the hydrogenase was 170 U (mg protein)−1 with methyl viologen and 833 U (mg protein)−1 with DMN as electron acceptors. Both holoenzymes showed molecular masses of 250 kDa. In SDS gels of active fractions, protein bands with apparent masses of 110 (SreA), 66 (HynL), 41 (HynS) and 29 kDa were present. Enriched hydrogenase fractions contained 14 μmol Fe and 2 μmol Ni (g protein)−1; in addition, 2·5 μmol Mo (g protein)−1 was found in the membrane fraction. Two overlapping genomic cosmid clones were sequenced, encoding a five-gene SR cluster (sre) including the 110 kDa subunit gene (sreA), and a 12-gene hydrogenase cluster (hyn) including the large and small subunit genes and genes encoding proteins required for the maturation of NiFe hydrogenases. A phylogenetic analysis of the SR amino acid sequence revealed that the protein belonged to the DMSO reductase family of molybdoenzymes and that the family showed a novel clustering. A model of sulfur respiration in Acidianus developed from the biochemical results and the data of the amino acid sequence comparisons is discussed.
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fusB is an allele of nadD, encoding nicotinate mononucleotide adenylyltransferase in Escherichia coli
More LessIsolation of the temperature-sensitive Escherichia coli mutant 72c has been described previously. The mutant allele was named fusB and causes a pleiotropic phenotype, the most striking features of which, besides temperature sensitivity, are the inability to grow on synthetic medium and supersensitivity to trimethoprim, an antibiotic that inhibits the C1 metabolism. This work shows that the fusB mutation is a frameshift mutation in the nadD gene that encodes nicotinate mononucleotide adenylyltransferase. The frameshift leads to a change of the last 10 amino acids and an addition of 17 amino acids. This lesion, renamed nadD72, leads to very little NAD+ and NADPH synthesis at the permissive temperature and essentially no synthesis at the non-permissive temperature. As a comparison, a new mutation in the nadD gene, with an amino acid change in the ATP-binding site, has been isolated. Its NAD+ synthesis is decreased at 30 °C but the level is still sufficient to support normal growth. At 42 °C, NAD+ synthesis is reduced further, which leads to temperature sensitivity on minimal medium. This mutation was designated nadD74. Thus, a small decrease in NAD+ levels affects ability to grow on minimal medium at 42 °C, while a large decrease leads to a more pleiotropic phenotype.
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Streptomyces lividans contains a minimal functional signal recognition particle that is involved in protein secretion
More LessThe bacterial version of the mammalian signal recognition particle (SRP) is well conserved and essential to all known bacteria. The genes for the Streptomyces lividans SRP components have been cloned and characterized. FtsY resembles the mammalian SRP receptor and the S. lividans SRP consists of Ffh, a homologue of the mammalian SRP54 protein, and scRNA, which is a small size RNA of 82 nt in length. Co-immunoprecipitation studies confirmed that Ffh and scRNA are probably the only components of the S. lividans SRP and that pre-agarase can co-immunoprecipitate with Ffh, suggesting that the SRP is involved in targeting secretory proteins.
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Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp.
More LessMicromonospora carbonacea var. africana ATCC 39149 contains a temperate bacteriophage, pMLP1, that is present both as a replicative element and integrated into the chromosome. Sequence analysis of a 4·4 kb KpnI fragment revealed pMLP1 att/int functions consisting of an integrase, an excisionase and the phage attachment site (attP). Plasmids pSPRH840 and pSPRH910, containing the pMLP1 att/int region, were introduced into Micromonospora spp. by conjugation from Escherichia coli. Sequence analysis of DNA flanking the integration site confirmed site-specific integration into a tRNAHis gene in the chromosome. The pMLP1 attP element and chromosomal bacterial attachment (attB) site contain a 24 bp region of sequence identity located at the 3′ end of the tRNA. Integration of pMLP1-based plasmids in M. carbonacea var. africana caused a loss of the pMLP1 phage. Placement of an additional attB site into the chromosome allowed integration of pSPRH840 into the alternate attB site. Plasmids containing the site-specific att/int functions of pMLP1 can be used to integrate genes into the chromosome.
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Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies
A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58·0 kDa. The enzyme had an isoelectric point of around pH 6·9. The optimum pH for enzyme activity was around 3·0 against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40 °C and stable up to 50 °C. The enzyme contained 8·6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p-phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. β-(3,4-Dihydroxyphenyl)alanine (l-DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes, was also oxidized by Lcc 2, and the oxidative product of l-dopa was identified as l-DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain.
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- Biodiversity And Evolution
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Multilocus sequence analysis of Porphyromonas gingivalis indicates frequent recombination
In this study, the genetic relationship of 19 Porphyromonas gingivalis isolates from patients with periodontitis was investigated by multilocus sequence analysis. Internal 400–600 bp DNA fragments of the 10 chromosomal genes ef-tu, ftsQ, hagB, gpdxJ, pepO, mcmA, dnaK, recA, pga and nah were amplified by PCR and sequenced. No two isolates were identical at all 10 loci. Phylogenetic analyses indicated a panmictic population structure of P. gingivalis. Split decomposition analysis, calculation of homoplasy ratios and analyses of clustered polymorphisms all indicate that recombination plays a major role in creating the genetic heterogeneity of P. gingivalis. A standardized index of association of 0·0898 indicates that the P. gingivalis genes analysed are close to linkage equilibrium.
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The variation of dTDP-l-rhamnose pathway genes in Vibrio cholerae
More LessThe genetic variation in the dTDP-l-rhamnose pathway genes (rmlA, rmlB, rmlC and rmlD) in Vibrio cholerae was investigated. The genes are part of the O antigen gene cluster and the aim was to study lateral gene transfer of O antigen gene clusters. The rml genes of an O6 strain were cloned using an Escherichia coli K-12 strain designed for selecting cloned rml genes. Thirty-three strains carrying the known rhamnose-containing O antigens were probed with O6-based rml gene probes, and 19 were positive with from one to all four of the gene probes. Nine rml gene sets from this group were sequenced and found to be in the order rmlBADC, at the 5′ end of the gene clusters. A gradient in the level of variation was observed, with highly similar sequences at the 5′ end rmlB gene, but very divergent and strain-specific sequences at the 3′ end of the rml gene set. The change in level of similarity varied in position, but was always abrupt and coincided with a change in GC content, indicating that the 5′ and 3′ parts are of different origin, and that recombination within rml genes has occurred. The rml gene sets of two of the strains that did not hybridize with any O6 rml gene probes were also cloned and sequenced. Both gene sets were in the middle of the O antigen gene cluster and were very divergent from each other and all other rml gene sets. This supports the hypothesis that presence of rml genes at the end of the O antigen gene cluster facilitates lateral gene transfer of rml-containing O antigen gene clusters in V. cholerae. The sequence relationships make it possible to identify sites of recombination and to distinguish DNA that has long been in V. cholerae and DNA that probably came into the species with the O antigen gene cluster.
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- Environmental Microbiology
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Analysis of genes of tetrahydrofolate-dependent metabolism from cultivated spirochaetes and the gut community of the termite Zootermopsis angusticollis
More LessThe hindguts of wood-feeding termites are the sites of intense, CO2-reductive acetogenesis. This activity profoundly influences host nutrition and methane emissions. Homoacetogens previously isolated from diverse termites comprised novel taxa belonging to two distinct bacterial phyla, Firmicutes and Spirochaetes. Little else is known about either the diversity or abundance of homoacetogenic species present in any given termite or the genetic details underlying CO2-reductive acetogenesis by Spirochaetes. A key enzyme of CO2-reductive acetogenesis is formyltetrahydrofolate synthetase (FTHFS). A previously designed primer set was used to amplify FTHFS genes from three isolated termite-gut spirochaetes. Sequencing DNA flanking the FTHFS gene of Treponema strain ZAS-2 revealed genes encoding two acetogenesis-related enzymes, methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolate dehydrogenase. Although termite-gut spirochaetes are only distantly related to clostridia at the ribosomal level, their tetrahydrofolate-dependent enzymes appear to be closely related. In contrast, homologous proteins identified in the non-homoacetogenic oral spirochaete Treponema denticola were only distantly related to those from clostridia and the termite-gut treponemes. Having demonstrated their utility with spirochaete pure cultures, the FTHFS primers were used to construct a 91-clone library from the termite-gut community DNA. From this, 19 DNA and eight amino acid FTHFS types were identified. Over 75 % of the retrieved clones formed a novel, coherent cluster with the FTHFS homologues obtained from the termite-gut treponemes. Thus, FTHFS gene diversity in the gut of the termite Zootermopsis angusticollis appears to be dominated by spirochaetes. The homoacetogenic capacity of termite-gut spirochaetes may have been acquired via lateral gene transfer from clostridia.
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A novel, fast-growing Borrelia sp. isolated from the hard tick Hyalomma aegyptium in Turkey
More LessA novel, fast-growing spirochaete was isolated from the hard tick Hyalomma aegyptium (family Ixodidae, subfamily Metastriata) using Barbour–Stoenner–Kelly (BSK) II medium. Tick samples were taken during the summer of 2000 from the Istanbul area in northwestern Turkey. Sixty-seven of 153 adults (44 %) and 72 of 185 nymphs (39 %) were infected with the novel spirochaete, whereas none of the 20 larvae examined were infected. The optimal growth temperature of the spirochaete in BSK II medium was 34–37 °C, and it could grow at 39 °C. Doubling times at 34 and 37 °C were 5·3 and 5·1 h, respectively. Six pure cultures of the spirochaete were obtained and characterized by microscopic observation, sequence analysis of the flagellin gene (flaB), SDS-PAGE and Western blotting. The spirochaete was morphologically similar to those of the genus Borrelia and contained a 41 kDa protein reactive with mAb H9724 specific to the flagellin of a Borrelia species. Polyclonal antibody raised to this spirochaete reacted with several antigen bands, whereas no bands were detected with Borrelia burgdorferi, Borrelia hermsii, Borrelia turicatae and Borrelia parkeri. The flaB sequences of the six isolates showed high similarity, with sequence similarity values ranging from 99·2 to 100 %; however, the similarity of the isolates' flaB sequences to those of the Lyme-disease-related Borrelia and relapsing-fever-associated Borrelia species was less than 90 %. These findings suggest that the unique spirochaete is a member of the genus Borrelia, and differs from previously described Borrelia species.
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- Genes And Genomes
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The complete genome sequence of the avian pathogen Mycoplasma gallisepticum strain Rlow
The complete genome of Mycoplasma gallisepticum strain Rlow has been sequenced. The genome is composed of 996 422 bp with an overall G+C content of 31 mol%. It contains 742 putative coding DNA sequences (CDSs), representing a 91 % coding density. Function has been assigned to 469 of the CDSs, while 150 encode conserved hypothetical proteins and 123 remain as unique hypothetical proteins. The genome contains two copies of the rRNA genes and 33 tRNA genes. The origin of replication has been localized based on sequence analysis in the region of the dnaA gene. The vlhA family (previously termed pMGA) contains 43 genes distributed among five loci containing 8, 2, 9, 12 and 12 genes. This family of genes constitutes 10·4 % (103 kb) of the total genome. Two CDSs were identified immediately downstream of gapA and crmA encoding proteins that share homology to cytadhesins GapA and CrmA. Based on motif analysis it is predicted that 80 genes encode lipoproteins and 149 proteins contain multiple transmembrane domains. The authors have identified 75 proteins putatively involved in transport of biomolecules, 12 transposases, and a number of potential virulence factors. The completion of this sequence has spawned multiple projects directed at defining the biological basis of M. gallisepticum.
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An ordered collection of expressed sequences from Cryphonectria parasitica and evidence of genomic microsynteny with Neurospora crassa and Magnaporthe grisea
More LessCryphonectria parasitica, the causative agent of chestnut blight, has proven to be a tractable experimental system for studying fungal pathogenesis. Moreover, the development of infectious cDNA clones of C. parasitica hypoviruses, capable of attenuating fungal virulence, has provided the opportunity to examine molecular aspects of fungal plant pathogenesis in the context of biological control. In order to establish a genomic base for future studies of C. parasitica, the authors have analysed a collection of expressed sequences. A mixed cDNA library was prepared from RNA isolated from wild-type (virus-free) and hypovirus-infected C. parasitica strains. Plasmid DNA was recovered from individual transformants and sequenced from the 5′ end of the insert. Contig analysis of the collected sequences revealed that they represented approximately 2200 individual ORFs. An assessment of functional diversity present in this collection was achieved by using the blast software utilities and the NCBI protein database. Candidate genes were identified with significant potential relevance to C. parasitica growth, development, pathogenesis and vegetative incompatibility. Additional investigations of a 12·9 kbp genomic region revealed microsynteny between C. parasitica and both Neurospora crassa and Magnaporthe grisea, two closely related fungi. These data represent the largest collection of sequence information currently available for C. parasitica and are now forming the basis of further studies using microarray analyses to determine global changes in transcription that occur in response to hypovirus infection.
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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)
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