- Volume 143, Issue 6, 1997
Volume 143, Issue 6, 1997
- Review Article
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- Microbiology Comment
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- Biochemistry
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Identification of a phenolic 3-O-methyltransferase in the lignin-degrading fungus Phanerochaete chrysosporium
More LessA methyl transferase enzyme catalysing the 3-O-methylation of isovanillic acid (3-hydroxy-4-methoxy!benzoic acid) by S-adenosylmethionine (SAM) was identified in Phanerochaete chrysosporium and purified. Gel filtration indicated an M r of 71000 and SDS-PAGE showed that the enzyme was composed of two sublimits of Mr approximately 36000. Substrate utilization studies demonstrated that the enzyme was highly specific, displaying an exclusive preference for the methylation of the 3-hydroxyl group of several substituted benzoic acids. 3-Hydroxybenzoic acids with a methoxyl or hydroxyl substituent in the 2 or 4 position were the best substrates with isovanillic and 3,4-dihydroxybenzoic acids showing the highest rates of methylation. The 3-O-methyltransferase enzyme was induced later in the growth cycle than the 4-O-methyltransferase previously isolated from this fungus, which is believed to have a role in the 4-O-methylation of lignin degradation products. However the function of this meta-specific enzyme, the first phenolic 3-O-methyltransferase isolated from a fungus, remains unclear. The combined activities of the 3- and 4-O-methyltransferase enzymes satisfactorily account for the pattern of SAM-dependent methylating activity shown by whole mycelia to phenolic substrates.
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- Bioenergetics And Transport
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Extrusion of benzoic acid in Saccharomyces cerevisiae by an energy-dependent mechanism
More LessWhen grown in the presence of benzoic acid, Saccharomyces cerevisiae was able to extrude [14C]benzoic acid when a pulse of glucose was given to preloaded cells. While octanoic, sorbic, hexanoic, salicylic, butyric and propionic acids were also inducers, ethanol and acetic acid were not. The mechanism of extrusion required energy and prior growth in the presence of the inducers. Diethylstilbestrol, an inhibitor of ATPases, prevented benzoic acid extrusion. Propionic acid was not actively extruded in cells adapted to either benzoic or propionic acid, behaving as an appropriate probe to measure intracellular pH. Even though the extrusion mechanism was active, benzoic acid entered the cells by a simple diffusion mechanism.
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- Biotechnology
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Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae: effects of insert position and host background
More LessThe potential of the major structural protein of type 1 fimbriae as a display system for heterologous sequences was tested. As a reporter-epitope, a heterologous sequence mimicking a neutralizing epitope of the cholera toxin B chain was inserted, in one or two copies, into four different positions in the fim gene. This was carried out by introduction of new restriction sites by PCR-mediated site-directed mutagenesis of fim in positions predicted to correspond to optimally surface-located regions of the subunit protein. Subsequently, the synthetic cholera-toxin-encoding DNA segment was inserted. Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested with respect to host background in three different Escherichia coli strains, i.e. an isogenic set of K-12 strains, differing in the presence of an indigenous fim gene cluster, as well as a wild-type isolate. Immunization of rabbits with purified chimeric fimbriae resulted in serum which specifically recognized cholera toxin B chain, confirming the utility of the employed strategy.
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- Environmental Microbiology
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Repression and inactivation of α-amylase in Thermomonospora species during growth on cellobiose
More LessThermophilic actinomycetes establish themselves as numerically dominant bacterial populations in selected high temperature environments by virtue of their exoenzymic ability to degrade the complex polysaccharides in thermogenic plant biomass. When Thermomonospora curvata and Thermomonospora fusca were grown on a mixture of cellulose and starch in mineral salts minimal medium, α-amylase was repressed via inhibition of maltose uptake by cellobiose. Addition of cellobiose to exponential phase cells growing on maltose or maltotriose triggered rapid degradation of extant amylase in the culture fluid of wild-type cells, but not in a protease-deficient mutant of T. fusca. A serine protease purified from T. fusca caused inactivation of the amylase in culture fluid of the mutant when added at a concentration approximating to that of the wild-type strain. The chelating agent, EDTA, accelerated inactivation by the protease, while the presence of calcium or amylase reaction products protected the amylase. Therefore, during growth in an environment containing multiple polysaccharides, these thermophiles control the levels of their extracellular depolymerizing enzymes via both inducer exclusion and proteolytic inactivation.
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The role of pseudolysogeny in bacteriophage-host interactions in a natural freshwater environment
More LessBacteriophages occur in high numbers in environmental ecosystems and are thus significant mediators of microbial survival and activities. However, interactions between microbial populations and phages in situ have been largely ignored. Current understanding of the process relies on studies performed with well-fed, laboratory-grown host bacteria. The purpose of the experiments reported here was to determine bacteriophage-host interactions under environmentally relevant conditions of nutrient limitation. These studies have revealed the importance of a phenomenon called pseudolysogeny in the maintenance of viral genetic material for extended periods of time in natural ecosystems. Pseudolysogeny is a form of phage-host cell interaction in which the nucleic acid of the phage resides within its starved host in an unstable, inactive state. It is hypothesized that pseudolysogeny occurs due to the cell's highly starved condition. In such cells, there is insufficient energy available for the phage to initiate genetic expression leading to either a true temperate response or to the lytic response. However, upon nutrient addition, the pseudolysogenic state is resolved, resulting in either the establishment of true lysogeny or the initiation of the lytic production of progeny virions. The pseudolysogenic state may explain the long-term survival of viruses in unfavourable environments in which the infective half-life of their virions is relatively short.
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Modulation of gene expression through chromosomal positioning in Escherichia coli
More LessVariations in expression of the nah genes of the NAH7 (naphthalene biodegradation) plasmid of Pseudomonas putida when placed in different chromosomal locations in Escherichia coli have been studied by employing a collection of hybrid mini-Tn5 transposons bearing lacZ fusions to the Psal promoter, along with the cognate regulatory gene nahR. Insertions of Psal-lacZ reporters in the proximity of the chromosomal origin of replication, oriC, increased accumulation of β-galactosidase in vivo. Position-dependent changes in expression of the reporter product could not be associated with local variations of the supercoiling in the DNA region, as revealed by probing the chromosome with mobile gyrB-lacZ elements. Such variations in β-galactosidase activity (and, therefore, the expression of catabolic genes) seemed, instead, to be linked to the increase in gene dosage associated with regions close to oriC, and not to local variations in chromosome structure. The tolerance of strains to the selection markers borne by the transposons also varied in parallel with the changes in LacZ levels. The role of chromosomal positioning as a mechanism for the outcome of adaptation phenotypes is discussed.
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- Genetics And Molecular Biology
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VlpA of Vibrio cholerae O1: the first bacterial member of the α2-microglobulin lipocalin superfamily
More LessWe have identified a gene, vlpA, which is closely linked to the mfrA,B locus associated with mannose-fucose-resistant haemagglutination. VlpA is an outer-membrane protein which can be labelled with [3H]palmitate and whose processing is globomycin-sensitive, suggesting that it is a lipoprotein. Homology searches revealed that VlpA belongs to the group of lipocalins of the α2-microglobulin superfamily which function as small hydrophobic molecule transporters, and is the first identified bacterial member of this group. Multiple copies of this gene are present in Vibrio cholerae O1 and O139 and Southern hybridization reveals a biotype-specific pattern of fragment sizes. Construction of strains capable of hyperproducing VlpA suggested that it is able to bind haemin with low affinity but this may be due to a simple hydrophobic interaction. Attempts to construct specific mutants in vlpA have been unsuccessful, presumably because of the multiple copies of vlpA genes and their linkage to the VCR element.
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Targeted gene-replacement mutagenesis of dcrA, encoding an oxygen sensor of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough
More LessA gene-replacement mutagenesis method has been developed for the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough and used to delete dcrA, encoding a potential oxygen or redox sensor with homology to the methyl-accepting chemotaxis proteins. A suicide plasmid, containing a cat-marked dcrA allele and a counter-selectable sacB marker was transferred from Escherichia coli S17-1 to D. vulgaris by conjugation. Following plasmid integration the desired dcrA deletion mutant (D. vulgaris F100) was obtained in media containing sucrose and chloramphenicol. Southern blot screening was required to distinguish D. vulgaris F100 from strains in which the sacB marker was inactivated by transposition of an endogenous IS element. No anaerotactic deficiency has so far been detected in D. vulgaris F100, which was found to be more resistant to inactivation by oxygen than the wild-type. Increased transcription of the rbo-rub operon, located immediately downstream from dcrA, was demonstrated by Northern blotting and may be the cause of this unusual phenotype, in view of the recent discovery that Rbo can complement the deleterious effects of superoxide dismutase deficiency in E. coli.
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Organization of methylamine utilization genes (mau) in ‘Methylobacillus flagellatum’ KT and analysis of mau mutants
More LessThe organization of genes involved in utilization of methylamine (mau genes) was studied in the obligate methylotroph ‘Methylobacillus flagellatum’ KT. Nine open reading frames were identified as corresponding to the genes mauFBEDAGLMN. In addition, an open reading frame (orf-1) encoding a polypeptide with unknown function was identified upstream of the mau gene cluster. Subclones of the ‘M. flagellatum’ KT gene cluster were used for complementation of a series of chemically induced mau mutants of ‘M. flagellatum’ KT. Mutants in mauF, mauB, mauEID, mauA, mauG, mauL and mauM were identified. Two mutants (mau-18 and mau-19) were not complemented by the known mau genes. Since none of the chemically induced mutants studied had a defect in orf-1 or mauN, insertion mutants in these genes were constructed. Phenotypically the mutants fell into three groups. The mauF, mauB, mauE/D, mauA, mauG, mauL and mauM mutants do not grow on methylamine as a source of carbon and lack methylamine dehydrogenase activity, but they synthesize both the large and the small subunit polypeptides albeit at different ratios. The mau-18 and mau-19 mutants do not grow on methylamine as a source of carbon, and lack both methylamine dehydrogenase activity and the methylamine dehydrogenase subunits. The orf-1 and mauN mutants grow on methylamine as a source of carbon and synthesize wild-type levels of methylamine dehydrogenase. It has been shown earlier that the product of the mauM gene is not required for synthesis of active methylamine dehydrogenase in Methylobacterium extorquens AM1 and Paracoccus denitrificans. However, MauM is required for synthesis of functional methylamine dehydrogenase in ‘M. flagellatum’.
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Construction and properties of aconitase mutants of Escherichia coli
More LessEscherichia coli contains two genes (acnA and acnB) encoding aconitase activities. An acnB mutant was engineered by replacing the chromosomal acnB gene by an internally deleted derivative containing a tet R cassette. An acnB double mutant was then made by transducing a previously constructed acnA::kan R mutation into the acnB::tet R strain. Western blotting confirmed that the AcnA and AcnB proteins were no longer produced by the corresponding mutants and PCR analysis showed that the chromosomal acnB gene had been replaced by the disrupted gene. Aerobic and anaerobic growth in glucose minimal medium were impaired but not abolished by the acnB mutation, indicating that the lesion is partially complemented by the acnA + gene, and growth was enhanced by glutamate. The acnAB double mutant would not grow on unsupplemented glucose minimal medium and although it responded to glutamate like a typical auxotroph under anaerobic conditions, under aerobic conditions no response to glutamate was observed before it was over-grown by ‘revertants’ lacking citrate synthase (acnAB gltA). The acnAB double mutant retained a low but significant aconitase activity (< 5% of wild-type), designated AcnC. Enzymological and regulatory studies with acn-lacZ fusions indicated that AcnB is the major aconitase, which is synthesized earlier in the growth cycle than AcnA, and subject to catabolite and anaerobic repression.
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Plasmid diversity in Chlamydia
More LessChlamydiae exhibit low interspecies DNA homology and plasmids from different chlamydial species can be readily distinguished by Southern blot analysis and restriction enzyme profiling. In contrast, available plasmid sequence data from within the species Chlamydia trachomatis indicate that plasmids from human isolates are highly conserved. To evaluate the nature and extent of plasmid variation, the complete nucleotide sequences were determined for novel plasmids from three diverse non-human chlamydial isolates: pCpA1 from avian Chlamydia psittaci (N352); pCpnE1 from equine Chlamydia pneumoniae (N16); and pMoPn from C. trachomatis mouse pneumonitis. Comparison of the sequence data did not identify an overall biological function for the plasmid but did reveal considerable sequence conservation (> 60%) and a remarkably consistent genomic arrangement comprising eight major ORFs and four 22 bp tandem repeats. The plasmid sequences were close to 7500 nucleotides in length (pCpA1, 7553 bp; pMoPn, 7502 bp) however the equine C. pneumoniae plasmid was smaller (7362 bp) than all other chlamydial plasmids. The reduced size of this plasmid was due to a single large deletion occurring within ORF 1; this potentially generates two smaller ORFs. The disruption of ORF 1 is the only significant variation identified amongst the chlamydial plasmids and could prove important for future vector development studies.
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Isolation of developmentally regulated genes from the edible mushroom Agaricus bisporus
More LessFrom a cDNA library, constructed from mushroom primordia, nine cDNAs were isolated which were either induced or specifically expressed during fruit body development and maturation of the basidiomycete Agaricus bisporus. These cDNAs varied in size from 372 to 1019 bp and hybridized to transcripts of 400-1600 nt. Four of the cDNAs were only expressed in the generative phase o: the life cycle while the other five cDNAs were strongly induced but had low steady-state mRNA levels in vegetatively grown mycelium of the hybrid strain Horst U1. An apparent full-length cDNA could be identified by sequence analysis and specified a putative protein homologous to the δ-subunit of the mitochondrial ATP synthase complex of Saccharomyces cerevisiae and Neurospora crassa. For one of the partial cDNAs, significant homology was found with a family of cell division control proteins, while another partial cDNA appeared to encode a cytochrome P450. All cDNAs, except the presumed cytochrome-P450-specifying cDNA (cypA), hybridized with single copy genes scattered over the Agaricus genome. For the cypA gene, the presence of several additional copies was shown by heterologous hybridizations. Based on changes in expression levels of the fruit-body-induced genes during development coinciding with alterations in morphological appearance of mushrooms, four stages of development were distinguished during growth and maturation of A. bisporus fruit bodies.
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β-Tubulin genes and the basts for benzimidazole sensitivity of the opportunistic fungus Cryptococcus neoformans
More LessThe basidiomycete Cryptococcus neoformans causes life-threatening infections in immunocompromised patients, and available chemotherapeutic agents are potentially toxic or have limited efficacy. in vitro, C. neoformans is very sensitive to selected benzimidazole compounds (e.g. albendazole), which act by disrupting microtubules through binding to the β-tubulin subunit. To understand the basis for this benzimidazole sensitivity, we have characterized C. neoformans β-tubulin genes and their expression. Analysis of PCR amplification products, genomic and cDNA clones and Southern blots identified two β-tubulin genes. TUB1 contains seven introns, including one that splits the start codon, and encodes a 447 amino acid protein with >80% identity to most other β-tubulins. A partial sequence of TUB2 revealed a higher density of introns and a considerably more divergent β-tubulin. The relative expression of TUB1 to TUB2 determined by reverse-transcription PCR was about 3:1, consistent with a more limited role for the TUB2 product. Comparisons of β-tubulin sequences from C. neoformans and from various benzimidazole-sensitive and -resistant organisms strongly suggest that the TUB1 product represents the primary benzimidazole target. This was supported by the identification of a His6 to Gin change in TUB1 from three independently isolated albendazole-resistant mutants.
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CHS2, a chitin synthase gene from the oomycete Saprolegnia monoica
More LessPCR was used to amplify fragments corresponding to the chitin synthase (CHS) genes from the Oomycetes Saprolegnia monoica, Phytophthora capsicum and Achlya ambisexualis, utilizing as primers, oligonucleotides designed from the conserved region of CHS genes of chitinous fungi. Chitin synthase homologues were found in the three cellulosic fungi. The chitin synthase 2 gene (CHS2) from S. monoica was cloned, sequenced and characterized. The amino acid sequence deduced from the CHS2 genomic DNA revealed several domains, corresponding to the catalytic domains and polypeptide signatures, of high identity with CHS genes from chitinous fungi. Existence of a CHS gene family in S. monoica was supported by the identification of two CHS sequences among the PCR products, the localization of CHS homologues on two chromosomes, and the detection of two transcripts in mycelia and protoplasts. Polyclonal anti-chitin synthase antibodies raised against the N-terminal and the neutral fragments of the CHS2 products revealed, respectively, two and four proteins in membrane fractions and a truncated active form in entrapped product. The overall comparison of the structure and organization of CHS genes indicates that in spite of their divergent evolution, Oomycetes and chitinous fungi have evolved with conserved chitin synthase systems.
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Interruption of the Streptococcus gordonii M5 sspA/sspB intergenic region by an insertion sequence related to IS1167 of Streptococcus pneumoniae
More LessStreptococcus gordonii M5 and DL1 each express two related adhesin polypeptides, SspA and SspB, which are members of the antigen I/II family of streptococcal surface proteins. The sspA and sspB genes are tandemly arranged in both strains, with sspA residing upstream of sspB. The genes are separated by approximately 400 nucleotides in S. gordonii DL1 and 1300 nucleotides in S. gordonii M5. The nucleotide sequence of the sspA/sspB intergenic region of strain M5 is reported and the difference in length compared to S. gordonii DL1 shown to arise from the presence of an insertion sequence, designated ISSg1, consisting of 1197 bp. The nucleotide sequence of ISSg1 is highly homologous to IS1167 of Streptococcus pneumoniae and is related to a lesser extent to other members of the IS1096 family of bacterial insertion sequences. It contains a single ORF of 1026 bp, encoding a putative transposase polypeptide of 342 amino acids. The deduced transposase sequence exhibits 93% identity with the transposase polypeptides encoded by IS1167. However, the S. gordonii protein lacks a 90 residue central domain that is present in the IS1167 transposase and in the transposase polypeptides encoded by the related IS elements. In addition, the organization of the inverted repeats flanking the transposase gene in S. gordonii differs from IS1167. Extension products generated from a sspB-specific primer indicated that transcription initiates within the intergenic region in both S. gordonii strains, suggesting that sspA and sspB are independently transcribed. Transcription appears to initiate 42 bases upstream of sspB in S. gordonii DL1 In contrast, sspB transcription in M5 initiates at least 125 bases upstream of sspB, in close proximity to the terminal inverted repeat of ISSg1. These results indicate that the sspB promoters of S. gordonii M5 and DL1 are not conserved and suggest that ISSg1 sequences may play a role in directing the expression of sspB in S. gordonii M5.
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The site-specific recombinase encoded by pinD in Shigella dysenteriae is due to the presence of a defective Mu prophage
More LessThe DNA inversion systems are made up of an invertible DNA segment and a site-specific recombinase gene. Five systems are known in prokaryotes: the Salmonella typhimurium H segment and hin gene (H-hin), phage Mu G-gin, phage P1 C-cin, Escherichia coli e14 P-pin, and Shigella sonnei B-pinB systems. In this report a site-specific recombinase (pinD) gene of Shigella dysenteriae was cloned and sequenced. pinD mediated inversion of five known segments at the same extent in E. coll. Although one inv sequence was identified, no invertible region was detected in a cloned fragment. The predicted amino acid sequences of PinD and three ORFs showed high homology to those of Gin and its flanking gene products. An ORF homologous to Mom of Mu conserved a functional activity to modify intracellular plasmid DNA. Southern analysis showed that the cloned fragment contains two homologous regions corresponding to the left and right ends of the Mu genome. Together these results indicated that the pinD gene in S. dysenteriae is derived from a Mu-like prophage.
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The aldA gene of Escherichia coli is under the control of at least three transcriptional regulators
More LessExpression studies on the aldA gene encoding aldehyde dehydrogenase in Escherichia coli showed induction by two types of molecule (hydroxyaldehydes and 2-oxoglutarate), carbon catabolite repression and respiration dependence. Promoter deletion analysis showed that the proximal operator, which includes inducer-regulator complex and catabolite repression protein (Crp) recognition sites, was necessary for induction by either type of inducer, and that full induction by aldehydes required the cooperation of distal operator sequences beyond position -119. Interactions of the regulator protein with the -59 to -6 fragment were shown by DNA mobility shift assays. Fusions of different deletions of the aldA promoter to lacZ indicated that a Crp site proximal to the transcriptional start point (tsp) was functional in the cAMP-dependent catabolite repression of this system, whereas a distal control site was likely to operate in a cAMP-independent catabolite repression. DNA mobility shift and footprint analyses showed that only the tsp proximal site was bound by pure Crp with a K d of 5.4 x 10-7 M. As shown by an Arc-defective strain, the aldA gene seems to be repressed by the Arc system under anaerobiosis, displaying its physiological full induction and activity in the presence of oxygen.
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- Pathogenicity And Medical Microbiology
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Escherichia coli LT enterotoxin subunit A demonstrates partial toxicity independent of the nicking around Arg192
A study was conducted into whether or not nicking of the A subunit of Escherichia coli LT enterotoxin at position Arg192 or its neighbouring amino acids Arg192 to The 195 is required for its toxicity. The toxic activity of mutants created by substitution or deletion at this position, which lacked ADP-ribosyltransferase activity in vitro, was not completely obliterated and cyclic AMP was partially induced in the target cells, showing that they still displayed enzymic activity in vivo. Moreover, although the A subunit possesses three potential sites for cleavage by furin, furin was not involved in the partial toxicity and cyclic AMP induction observed. These data suggest that target cells have a nick mechanism that operates at sites other than those around Arg192 or those recognized by furin, which generates an active fragment by processing the A subunit after toxin binding to the cell membrane.
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Adaptation of proteases and carbohydrases of saprophytic, phytopathogenic and entomopathogenic fungi to the requirements of their ecological niches
More LessThe abilities of isolates of saprophytes (Neurospora crassa, Aspergillus nidulans), an opportunistic human pathogen (Aspergillus fumigatus), an opportunistic insect pathogen (Aspergillus flavus), plant pathogens (Verticillium albo-atrum, Verticillium dahliae, Nectria haematococca), a mushroom pathogen (Verticillium fungicola) and entomopathogens (Verticillium lecanii, Beauveria bassiana, Metarhizium anisopliae) to utilize plant cell walls and insect cuticle components in different nutrient media were compared. The pathogens showed enzymic adaptation to the polymers present in the integuments of their particular hosts. Thus, the plant pathogens produced high levels of enzymes capable of degrading pectic polysaccharides, cellulose and xylan, as well as a cutinase substrate, but secreted little or no chitinase and showed no proteolytic activity against elastin and mucin. The entomopathogens and V. fungicola degraded a broad spectrum of proteins (including elastin and mucin) but, except for chitinase, cellulase (V. lecanii and V. fungicola only) and cutinase (B. bassiana only), produced very low levels of polysaccharidases. The saprophytes (Neu. crassa and A. nidulans) and the opportunistic pathogens (A. fumigatus and A. flavus) produced the broadest spectrum of protein and polysaccharide degrading enzymes, indicative of their less specialized nutritional status. V. lecanii and V. albo-atrum were compared in more detail to identify factors that distinguish plant and insect pathogens. V. albo-atrum, but not V. lecanii, grew well on different plant cell wall components. The major class of proteases produced in different media by isolates of V. albo-atrum and V. dahliae were broad spectrum basic (pl > 10) trypsins which degrade Z-AA-AA-Arg-NA substrates (Z, benzoyl; AA, various amino acids; NA, nitroanilide), hide protein azure and insect (Manduca sexta) cuticles. Analogous peptidases were produced by isolates of V. lecanii and V. fungicola but they were specific for Z-Phe-Val-Arg-NA. V. albo-atrum and V. dahliae also produced low levels of neutral (pl ca 7) and basic (pl ca 9.5) subtilisin-like proteases active against a chymotrypsin substrate (Succinyl-Ala2-Pro-Phe-NA) and insect cuticle. In contrast, subtilisins comprised the major protease component secreted by V. lecanii and V. fungicola. Both V. lecanii and V. albo-atrum produced the highest levels of subtilisin and trypsin-like activities during growth on collagen or insect cuticle. Results are discussed in terms of the adaptation of fungi to the requirements of their ecological niches
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- Physiology And Growth
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>Filamentous growth of the budding yeast Saccharomyces cerevisiae induced by overexpression of the WH12 gene
More LessThe WH12 gene of the budding yeast Saccharomyces cerevisiae is required for the arrest of cell proliferation upon nutrient exhaustion: whi2 mutants carry on dividing and in the absence of growth become abnormally SmaIl. It is reported here that overexpression of Whi2 from the GAL1 promoter results in filamentous growth - cells fail to complete cytokinesis, the budding pattern changes from axial to polar, cells become elongated and cell size increases threefold. In many ways, these filaments resemble the pseudohyphae which result from nitrogen-limited growth and the filaments seen during the invasive growth of haploids. However, Whi2-induced filament formation is reduced, but not blocked, by mutations in STE7, STE12 or STE20 which do block pseudohypha formation. Furthermore, pseudohypha formation can still occur in a diploid in which both copies of the WH12 gene have been deleted. Thus Whi2-induced filament formation and pseudohypha formation must come about through the action of different pathways. Despite this, a mutation in the STE11 gene, which is required for pseudohypha formation, does block Whi2-induced filament formation. Concanavalin A pulse-chase experiments show that new cell wall material is incorporated only into the tips of the apical cells. An extragenic suppressor of the whi2 allele also results in filamentous growth. These results suggest that Whi2 negatively regulates a function required for the budding mode of cell proliferation including cytokinesis. This function is defined wholly or in part by the fswl allele.
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An important role for glutathione and γ-glutamyltranspeptidase in the supply of growth requirements during nitrogen starvation of the yeast Saccharomyces cerevisiae
More LessWhen the yeast Saccharomyces cerevisiae Σ1278b was starved for nitrogen, the total glutathione (GSH) pool increased from 7 to 17 nmol (mg dry wt)-1 during the first 2 h and then declined. More than 90% of the total GSH shifted towards the central vacuole during this time. This transient stimulation was not observed in the presence of buthionine-(S,R)-sulphoximine (BSO), a specific transition-state-analogue inhibitor of γ-glutamylcysteine synthase (γ-GCS), nor in a mutant strain deficient in this enzyme. γ-Glutamyltranspeptidase (γ-GT), a vacuolar enzyme responsible for the initial step of GSH degradation, was derepressed during nitrogen starvation. This mechanism can apparently enable the starved yeast cell to use the constituent amino acids from GSH which accumulate in the vacuole to satisfy its growth requirements for nitrogen.
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Effects of various types of stress on the metabolism of reserve carbohydrates in Saccharomyces cerevisiae: genetic evidence for a stress-induced recycling of glycogen and trehalose
More LessIt is well known that glycogen and trehalose accumulate in yeast under nutrient starvation or entering into the stationary phase of growth, and that high levels of trehalose are found in heat-shocked cells. However, effects of various types of stress on trehalose, and especially on glycogen, are poorly documented. Taking into account that almost all genes encoding the enzymes involved in the metabolism of these two reserve carbohydrates contain between one and several copies of the stress-responsive element (STRE), an investigation was made of the possibility of a link between the potential transcriptional induction of these genes and the accumulation of glycogen and trehalose under different stress conditions. Using transcriptional fusions, it was found that all these genes were induced in a similar fashion, although to various extents, by temperature, osmotic and oxidative stresses. Experiments performed with an msn2/msn4 double mutant proved that the transcriptional induction of the genes encoding glycogen synthase (GSY2) and trehalose-6-phosphate synthase (TPS1) was needed for the small increase in glycogen and trehalose upon exposure to a mild heat stress and salt shock. However, the extent of transcriptional activation of these genes upon stresses in wild-type strains was not correlated with a proportional rise in either glycogen or trehalose. The major explanation for this lack of correlation comes from the fact that genes encoding the enzymes of the biosynthetic and of the biodegradative pathways were almost equally induced. Hence, trehalose and glycogen accumulated to much higher levels in cells lacking neutral trehalase or glycogen phosphorylase exposed to stress conditions, which suggested that one of the major effects of stress in yeast is to induce a wasteful expenditure of energy by increasing the recycling of these molecules. We also found that transcriptional induction of STRE-controlled genes was abolished at temperatures above 40 °C, while induction was still observed for a heat-shock-element-regulated gene. Remarkably, trehalose accumulated to very high levels under this condition. This can be explained by a stimulation of trehalose synthase and inhibition of trehalase by high temperature.
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Glucose-dependent, cAMP-mediated ATP efflux from Saccharomyces cerevisiae
More LessExtracellular ATP plays an important role in the physiology of multicellular organisms; however, it is unknown whether unicellular organisms such as yeast also release ATP extracellularly. Experiments are described here which show that Saccharomyces cerevisiae releases ATP to the extracellular fluid. This efflux required glucose and the rate was increased dramatically by the proton ionophores nigericin, monensin, carbonyl cyanide m-chlorophenylhydrazone and carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone; ATP efflux was also increased by the plasma membrane proton pump inhibitor diethylstilbestrol. The increase in the concentration of extracellular ATP was not due to cell lysis or general disruption of plasma membrane integrity as measured by colony-forming and methylene-blue-staining assays. ATP efflux was strictly correlated with a rise in intracellular cAMP; therefore, the cAMP pathway is likely to be involved in triggering ATP efflux. These results demonstrate that yeast cells release ATP in a regulated manner.
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The relationship between external glucose concentration and cAMP levels inside Escherichia coli: implications for models of phosphotransferase-mediated regulation of adenylate cyclase
More LessThe concentration of glucose in the medium influences the regulation of cAMP levels in Escherichia coli. Growth in minimal medium with micromolar glucose results in 8- to 10-fold higher intracellular cAMP concentrations than observed during growth with excess glucose. Current models would suggest that the difference in cAMP levels between glucose-rich and glucose-limited states is due to altered transport flux through the phosphoenolpyruvate : glucose phosphotransferase system (PTS), which in turn controls adenylate cyclase. A consequence of this model is that cAMP levels should be inversely related to the saturation of the PTS transporter. To test this hypothesis, the relationship between external glucose concentration and cAMP levels inside E. coli were investigated in detail, both through direct cAMP assay and indirectly through measurement of expression of cAMP-regulated genes. Responses were followed in batch, dialysis and glucose-limited continuous culture. A sharp rise in intracellular cAMP occurred when the nutrient concentration in minimal medium dropped to approximately 0∙3 mM glucose. Likewise, addition of >0∙3 mM glucose, but not <0∙3 mM glucose, sharply reduced the intracellular cAMP level of starving bacteria. There was no striking shift in growth rate or [14C]glucose assimilation in bacteria passing through the 0∙5 to 0∙3 mM concentration threshold influencing cAMP levels, suggesting that neither metabolic flux nor transporter saturation influenced the sensing of nutrient levels. The (IIA/IIBC)GIc PTS is 96–97% saturated at 0∙3 mM glucose so these results are not easily reconcilable with current models of cAMP regulation. Aside from the transition in cAMP levels initiated above 0∙3 mM, a second shift occurred below 1 μM glucose. Approaching starvation, well below saturation of the PTS, cAMP levels either increased or decreased depending on unknown factors that differ between common E. coli K-12 strains.
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Anaerobic taurine oxidation: a novel reaction by a nitrate-reducing Alcaligenes sp.
More LessEnrichment cultures were prepared under strictly anoxic conditions in medium representing fresh water and containing an organosulfonate as electron donor and carbon source, and nitrate as electron acceptor. The inoculum was from the anaerobic digestor of two communal sewage works. The natural organosulfonates 2-aminoethanesulfonate (taurine), dl-2-amino-3-sulfopropionate (cysteate) and 2-hydroxyethanesulfonate (isethionate) all gave positive enrichments, whereas unsubstituted alkanesulfonates, such as methanesulfonate and arenesulfonates, gave no enrichment. Two representative enrichments were used to obtain pure cultures, and strains NKNTAU (utilizing taurine) and NKNIS (utilizing isethionate) were isolated. Strain NKNTAU was examined in detail. Out of 18 tested organosulfonates, it utilized only one, taurine, and was identified as a novel Alcaligenes sp., a facultatively anaerobic bacterium. Carbon from taurine was converted to cell material and carbon dioxide. The amino group was released as ammonium ion and the sulfonate moiety was recovered as sulfate. Nitrate was reduced to nitrogen gas.
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Xanthobacter flavus employs a single triosephosphate isomerase for heterotrophic and autotrophic metabolism
More LessThe expression of the cbb and gap–pgk operons of Xanthobacter flavus encoding enzymes of the Calvin cycle is regulated by the transcriptional regulator CbbR. In order to identify other genes involved in the regulation of these operons, a mutant was isolated with a lowered activity of a fusion between the promoter of the cbb operon and the reporter gene lacZ. This mutant was unable to grow autotrophically and had a reduced growth rate of medium supplemented with gluconate or succinate. The regulation of the gap–pgk operon in the mutant was indistinguishable from the wild-type strain, but induction of the cbb operon upon transition to autotrophic growth conditions was delayed. Complementation of the mutant with a genomic library of X. flavus resulted in the isolation of a 1∙1 kb ApaI fragment which restored autotrophic growth of the mutant. One open reading frame (ORF) was present on the ApaI fragment, which could encode a protein highly similar to triosephosphate isomerase proteins from other bacteria. Cell extracts of the mutant grown under glycolytic or gluconeogenic conditions had severely reduced triosephosphate isomerase activities. The ORF was therefore identified as tpi, encoding triosephosphate isomerase. The tpi gene is not linked to the previously identified operons encoding Calvin cycle enzymes and therefore represents a third transcriptional unit required for autotrophic metabolism.
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The thioredoxin reductase system of mycoplasmas
More LessRepresentative species of the Mollicutes possess a thioredoxin reductase system (NTS) composed of a low-molecular-mass thioredoxin (TRX) and NADPH-binding thioredoxin reductase (NTR). The TRXs of Mycoplasma pneumoniae and M. capricolum have molecular masses of 11.2 and 12 kDa, respectively, and are stable at 90 °C for 10 min. Both TRXs reacted with monospecific polyclonal antibodies generated against the Bacillus subtilis TRX, but not with anti-Escherichia coli TRX antisera. The M. capricolum and M. pneumoniae NTRs were partially purified and were found to be active with the homologous TRX, but not with the TRX of B. subtilis or E. coli. The NTS activity had an optimal pH of 6.5-7.5 and was dependent on NADPH as an electron donor, a requirement which could not be fulfilled by NADH. The genes encoding the TRX and NTR (trxA and trxB) of M. pneumoniae were cloned and sequenced. The comparative analysis of the predicted amino acid sequence of trxA showed that the 11.2 kDa protein (102 aa) shared 26-68% sequence similarity with products of other known trxA genes and contained the conserved active site Cys-Gly-Pro-Cys. The predicted amino acid sequence of trxB contained 315 residues with a conserved NADPH binding domain and FAD binding domains I and II. The cysteine dithiol redox active region had isoleucine rather than threonine at the active site, as compared with other NTRs. The high activity of the NTS in mycoplasmas suggests that mycoplasmas may have evolved the NTS to protect themselves from the consequences of their self-generated oxidative challenge.
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Mutations to antibiotic resistance occur during the stationary phase in Lactobacillus plantarum ATCC 8014
More LessWhen Lactobacillus plantarum ATCC 8014 was maintained in LCM broth (which consists of buffered tryptone and is sufficient to support the growth of some species of Lactobacillus ) for long periods (120 d), viable bacteria persisted. Rifampicin-, streptomycin- and sodium-fusidate-resistant mutants were recovered from parallel LCM broth cultures following a stochastic pattern. Individual cultures appeared to yield mutants intermittently. One culture in particular yielded rifampicin-resistant colonies at a frequency of 1 in 100 viable bacteria after 20 d incubation and these persisted until the experiment was terminated at 115 d. In a separate experiment two parallel cultures yielded mutants resistant to low concentrations of streptomycin at a similar frequency. Using a chemostat it was shown that in continuous culture in LCM at slow growth rates the highest frequency of recovery of antibiotic-resistant mutants was achieved when the bacteria exhibited doubling times of 90 h or greater. The frequency of recovery of mutants was as high as 1 in 1000 viable bacteria. Thus, mutations to antibiotic resistance in L. plantarum ATCC 8014 can take place in the absence of measurable cell division. The data are consistent with the notion that populations of starved bacteria in stationary phase can be genetically dynamic.
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Regulation of exopolysaccharide production in Rhizobium leguminosarum biovar viciae WSM710 involves exoR
More LessA mildly acid-sensitive mutant of Rhizobium leguminosarum bv. viciae WSM710 (WR6-35) produced colonies which were more mucoid in phenotype than the wild-type. Strain WR6-35 contained a single copy of Tn5 and the observed mucoid phenotype, acid sensitivity and Tn5-induced kanamycin resistance were 100% co-transducible using phage RL38. WR6-35 produced threefold more exopolysaccharide (EPS) than the wild-type in minimal medium devoid of a nitrogen source. EPS produced by the mutant and the wild-type was identical as determined by proton NMR spectra. An EcoRI rhizobial fragment containing Tn5 and flanking rhizobial sequences was cloned from the mutant, restriction mapped and sequenced. There was extensive similarity between the ORF disrupted by Tn5 in R. leguminosarum bv. viciae WR6-35 and the exoR gene of Rhizobium (Sinorhizobium) meliloti Rm1021 (71-3% identity over 892 bp). At the protein level there was 70% identity and 93-3% similarity over 267 amino acids with the ExoR protein of R. meliloti Rm1021. Hydrophilicity profiles of the two proteins from these two rhizobia are superimposable. This gene in R. leguminosarum bv. viciae was thus designated exoR. The data suggest that Tn5 has disrupted a regulatory gene encoding a protein that negatively modulates EPS biosynthesis in R. leguminosarum bv. viciae WSM710. Despite earlier suggestions that EPS production and acid tolerance might be positively correlated, disruption of exoR in either R. leguminosarum bv. viciae or R. meliloti and its associated overproduction of EPS does not result in a more acid-tolerant phenotype than the wild-type when cultures are screened on conventional laboratory agar.
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A Neurospora crassa mutant altered in the regulation of L-amino acid oxidase
More LessThe isolation and characterization of a Neurospora crassa mutant altered in l-amino oxidase regulation is reported. The previously isolated gln-1bR8 strain, which only synthesizes the glutamine synthetase α monomer and lacks the β monomer, was used as parental strain. A mutant derivative of strain was selected for its ability to grow on minimal medium in the presence of DL-methionine-SR-sulfoximine (MSO), an inhibitor of glutamine synthetase activity. This gln-1bR8;MSO R mutant overcame the inhibitory effect of MSO by increasing the activity of L-amino acid oxidase, an enzyme capable of degrading this compound. In contrast with the wild-type strain, the L-amino acid oxidase of the MSOR mutant was resistant to glutamine repression; in fact, it was induced by this amino acid but repressed by ammonium. This mutant is different from other nitrogen regulatory N. crassa mutants reported and is only altered in the regulation of L-amino acid oxidase. The MSOR mutation is epistatic to nit-2 since the nit2;MSO R double mutant regulated the L-amino acid oxidase in the same way as the MSO R single mutant.
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Metabolic regulation of Irp gene expression in Escherichia coli K-12
More LessExpression of the Irp gene is regulated in part by the nutrients available to the cell, and is decreased in rich medium, in glucose minimal media enriched with amino acids, and in minimal medium with alternative carbon sources, such as acetate and succinate. When Lrp production is increased in a given medium, expression of its target genes is also increased. However, when the medium is changed from glucose to acetate, the response of the target genes is governed by many factors.
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- Plant-Microbe Interactions
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The symbiotic phenotypes of exopolysaccharide-defective mutants of Rhizobium sp. strain TAL1145 do not differ on determinate- and indeterminate-nodulating tree legumes
More LessThree classes of exopolysaccharide (EPS) defective mutants were isolated by Tn3Hogus -insertion mutagenesis of Rhizobium sp. strain TAL1145, which nodulates tree legumes. The class I and class III mutants produced 10-22% of the EPS produced by TAL1145 and appeared partially mucoid while the class II mutants formed small, opaque and non-mucoid colonies. Size-fractionation of the soluble EPSs made by these mutants in the culture supernatant indicated that the class I and the class III mutants produced reduced levels of both highland low-molecular-mass EPSs while the class II mutants lacked both these EPSs but produced a small amount of a medium-molecular-mass anthrone-reactive EPS. The succinyl and acetyl substituents observed in the TAL1145 EPS were absent in the EPS of the class II mutants. When examined under UV, the class I and class III mutants grown on Calcofluor-containing YEM agar showed dim blue fluorescence, compared to the bright blue fluorescence of the wild-type strain, whereas the class II mutants did not fluoresce. While the dim blue fluorescence of the class III mutants changed to yellow-green after 10 d, the fluorescence of the class I mutants did not change after prolonged incubation. Unlike the EPS-defective mutants of other rhizobia, these mutants did not show different symbiotic phenotypes on determinate- and indeterminate-nodulating tree legumes. The class I and the class III mutants formed small ineffective nodules on both types of legumes whereas the class II mutants formed normal nitrogen-fixing nodules on both types. The genes disrupted in the class I and class III mutants form a single complementation group while those disrupted in the class II mutants constitute another. All the three classes of EPS-defective mutants were located within a 10.8 kb region and complemented by two overlapping cosmids.
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- Systematics
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The genetic structure of Escherichia coli populations in feral house mice
More LessEscherichia coli was isolated from feral house mice (Mus domesticus) during the course of a mouse plague in the state of Victoria, Australia. Two farms were sampled over a period of 7 months and a total of 447 isolates were collected. The isolates were characterized using the techniques of randomly amplified polymorphic DNA and multi-locus enzyme electrophoresis. The mean genetic diversity of this E. coli population (H = 0.24) was found to be substantially lower than the diversity of an E. coli population reported elsewhere for a single human host. Analysis of the allozyme data revealed that there were significant differences in the relative abundance of genotypes between the two localities sampled and among sample dates. Overall, however, spatial and temporal effects accounted for less than 5% of the genotypic diversity. Allele frequencies and the relative abundance of the more common genotypes did not differ between male and female hosts. The number of genotypes and genotype diversity increased as the age of the host increased, suggesting that the mice are continuing to acquire new E. coli clones throughout their life. The frequency of some alleles changed with respect to host age, which indicates that clone acquisition may not be a random process. It is argued that the low level of genetic diversity observed in this population of E. coli reflects the boom and bust nature of mouse population density in this region of Australia.
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- Genome Analysis
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The Bacillus subtilis 168 chromosome from sspE to katA
More LessWe have cloned and sequenced a 24.5 kb region of the Bacillus subtilis 168 chromosome spanning the sspE and katA genes. The region contains a ribosomal RNA operon, rrnD, a tRNA gene set, trnD and 17 ORFs, 16 with putative ribosome-binding sites. Four of the ORFs (ORF2, ORF14, ORF16 and ORF17) match to known B. subtilis genes (sspE, thiA, senS and katA). Eight of the remaining ORF products show similarities with proteins present in the databases, including an ATP-binding transport protein, a glutamate-1-semialdehyde aminotransferase, a thiol-specific antioxidant protein, a mitomycin radical oxidase and a ferric uptake regulation protein.
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Nucleotide sequence and analysis of the phoB-rrnE-groESL region of the Bacillus subtilis chromosome
A 36 kb sequence of the phoB-rrnE-groESL region of the Bacillus subtilis chromosome at around 55° has been determined. The sequenced region contains 36 ORFs including the phoB and groESL genes, and the whole rrnE operon. The phoB gene is transcribed in the direction opposite to that of chromosome replication, while most ORFs, including groESL and the rrnE operon, are transcribed in the same direction. Two newly identified tRNA genes upstream of the rrnE operon were those for Arg-tRNA and Gly-tRNA. The sequenced region contains an operon consisting of genes for degradation and uptake of mannan. The rrnE operon and its downstream ORFs are well conserved among Mycoplasma genitalium, Haemophilus influenzae, Synechocystis sp. and Methanococcus jannaschii. σH consensus sequences are present in the promoter regions of three ORFs, including groESL.
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