- Volume 145, Issue 11, 1999
Volume 145, Issue 11, 1999
- Microbiology Comment
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- Biochemistry
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Novel Helicobacter pylori α1,2-fucosyltransferase, a key enzyme in the synthesis of Lewis antigens
More LessHelicobacter pylori lipopolysaccharides (LPS) contain complex carbohydrates known as Lewis antigens which may contribute to the pathogenesis and adaptation of the bacterium. Involved in the biosynthesis of Lewis antigens is an α1,2-fucosyltransferase (FucT) that adds fucose to the terminal βGal unit of the O-chain of LPS. Recently, the H. pylori (Hp) α1,2-FucT-encoding gene (fucT2) was cloned and analysed in detail. However, due to the low level of expression and instability of the protein, its enzymic activity was not demonstrated. In this study, the Hp fucT2 gene was successfully overexpressed in Escherichia coli. Sufficient amounts of the protein were obtained which revealed α1,2-fucosyltransferase activity to be associated with the protein. A series of substrates were chosen to examine the acceptor specificity of Hp α1,2-FucT, and the enzyme reaction products were identified by capillary electrophoresis. In contrast to the normal mammalian α1,2-FucT (H or Se enzyme), Hp α1,2-FucT prefers to use Lewis X [βGal1-4(αFuc1-3)βGlcNAc] rather than LacNAc [βGal1-4βGlcNAc] as a substrate, suggesting that H. pylori uses a novel pathway (via Lewis X) to synthesize Lewis Y. Hp α1,2-FucT also acts on type 1 acceptor [βGal1-3βGlcNAc] and Lewis a [βGal1-3(αFuc1–4)βGlcNAc], which provides H. pylori with the potential to synthesize H type 1 and Lewis b epitopes. The ability to transfer fucose to a monofucosylated substrate (Lewis X or Lewis a) makes Hp α1,2-FucT distinct from normal mammalian α1,2-FucT.
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The oxygenase component of the2-aminobenzenesulfonate dioxygenase system from Alcaligenes sp. strain O-1
More LessThe GenBank accession number for the sequence reported in this paper is AF109074.
Growth of Alcaligenes sp. strain O-1 with 2-aminobenzenesulfonate (ABS; orthanilate) as sole source of carbon and energy requires expression of the soluble, multicomponent 2-aminobenzenesulfonate 2,3-dioxygenase system (deaminating) (ABSDOS) which is plasmid-encoded. ABSDOS was separated by anion-exchange chromatography to yield a flavin-dependent reductase component and an iron-dependent oxygenase component. The oxygenase component was purified to about 98% homogeneity and an α2β2 subunit structure was deduced from the molecular masses of 134, 45 and 16 kDa for the native complex, and the α and β subunits, respectively. Analysis of the amount of acid labile sulfur and total iron, and the UV spectrum of the purified oxygenase component indicated one [2Fe–2S] Rieske centre per α subunit. The inhibition of activity by the iron-specific chelator o-phenanthroline indicated the presence of an additional iron-binding site. Recovery of active protein required strictly anoxic conditions during all purification steps. The FAD-containing reductase could not be purified. ABSDOS oxygenated nine sulfonated compounds; no oxygen uptake was detected with carboxylated aromatic compounds or with aliphatic sulfonated compounds. K m values of 29, 18 and 108 μM and V max values of 140, 110 and 72 pkat for ABS, benzenesulfonate and 4-toluenesulfonate, respectively, were observed. The N-terminal amino acid sequences of the α- and β-subunits of the oxygenase component allowed PCR primers to be deduced and the DNA sequence of the α-subunit was thereafter determined. Both redox centres were detected in the deduced amino acid sequence. Sequence data and biochemical properties of the enzyme system indicate a novel member of the class IB ring-hydroxylating dioxygenases.
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Anaerobic toluene-catabolic pathway in denitrifying Thauera aromatica: activation and β-oxidation of the first intermediate, (R)-(+)-benzylsuccinate
More LessAnaerobic catabolism of toluene is initiated by addition of the methyl group of toluene to the double bond of a fumarate cosubstrate to yield the first intermediate, benzylsuccinate. This reaction is catalysed by the glycyl-radical enzyme benzylsuccinate synthase, as shown for the denitrifying bacterium Thauera aromatica. Benzylsuccinate is further oxidized to benzoyl-CoA, the central intermediate of anaerobic degradation of aromatic compounds. The authors show here by experiments with cell extracts of toluene-grown T. aromatica that the pathway of benzylsuccinate oxidation requires activation of the free acid to a CoA-thioester, catalysed by a toluene-induced, reversible succinyl-CoA-dependent CoA-transferase. The product of the CoA-transferase reaction, benzylsuccinyl-CoA, is oxidized to benzoyl-CoA and succinyl-CoA in extracts of toluene-grown cells, adding proof to the proposed anaerobic toluene-catabolic pathway. The stereochemical preferences of the enzymes catalysing formation and activation of benzylsuccinate have been analysed. Benzylsuccinate synthase was found to produce exclusively (R)-(+)-benzylsuccinate, although the proposed reaction mechanism of this enzyme proceeds via radical intermediates. In accordance, the reaction of succinyl-CoA:benzylsuccinate CoA-transferase is also specific for (R)-(+)-benzylsuccinate and does not proceed with the (S)-(−)-enantiomer.
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- Biotechnology
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Organization of threonine biosynthesis genes from the obligate methylotroph Methylobacillus flagellatus
More LessThe GenBank accession numbers for the aspC-hom-thrC-thyA gene cluster and thrB gene sequences from M. flagellatus reported in this paper are L78665 and L78666, respectively.
The genes encoding aspartate kinase (ask), homoserine dehydrogenase (hom), homoserine kinase (thrB) and threonine synthase (thrC) from the obligate methylotroph Methylobacillus flagellatus were cloned. In maxicells hom and thrC directed synthesis of 51 and 48 kDa polypeptides, respectively. The hom, thrB and thrC genes and adjacent DNA areas were sequenced. Of the threonine biosynthesis genes, only hom and thrC were tightly linked in the order hom-thrC. The gene for thymidylate synthase (thyA) followed thrC and the gene for aspartate aminotransferase (aspC) preceded hom. All four genes (aspC-hom-thrC-thyA) were transcribed in the same direction. mRNA analysis indicated that hom-thrC are apparently transcribed in one 7·5 kb transcript in M. flagellatus. Promoter analysis showed the presence of a functional promoter between aspC and hom. No functional promoter was found to be associated with the DNA stretch between hom and thrC. The thrB gene encoded an unusual type of homoserine kinase and was not linked to other threonine biosynthesis genes.
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- Development And Structure
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Actin-related proteins in Actinobacillus pleuropneumoniae and their interactions with actin-binding proteins
A group of prokaryotic actin-related proteins (PARP) with an M r of 43000 was detected in Actinobacillus pleuropneumoniae. These proteins were enriched by a depolymerization/polymerization cycle, under similar conditions to those used to polymerize muscle actin, and purified by affinity chromatography on a DNase I-Sepharose column. Three isoforms of A. pleuropneumoniae PARP (Ap-PARP) with pI values of 5·8, 6·15 and 6·2 were detected. Ap-PARP were recognized by four different anti-actin antibodies (one anti-muscle and three anti-cytoplasmic isoforms). Ap-PARP were also recognized by antibodies against Anabaena variabilis PARP (Av-PARP) and against actin-binding proteins such as α-actinin and spectrin, and also by a monoclonal antibody against heat-shock cognate protein 70 (Hsc70). Specific binding of phalloidin to Ap-PARP was detected both in permeabilized cells and in vitro. Purified Ap-PARP can polymerize under similar conditions to those required for skeletal muscle actin polymerization and the filaments formed appear to be decorated with myosin subfragment-1 (S1) as observed by transmission electron microscopy. The amino acid composition of Ap-PARP revealed more similarities to muscle γ-actin and the cytoplasmic β-actin isoform than to eukaryotic actin-related proteins.
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- Environmental Microbiology
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Microbial diversity in marine sediments from Sagami Bay and Tokyo Bay, Japan, as determined by 16S rRNA gene analysis
More LessThe DDBJ accession numbers for the sequences reported in this paper are AB022607–AB022642.
16S rDNA clone libraries were analysed to investigate the microbial diversity in marine sediments from Sagami Bay (stations SA, water depth of 1159 m, and SB, 1516 m) and Tokyo Bay (station TK, 43 m). A total of 197 clones was examined by amplified rDNA restriction analysis (ARDRA) using three four-base-specific restriction enzymes (HhaI, RsaI and HaeIII). In SA, 57 RFLP types were detected from 77 clones. In SB, 17 RFLP types were detected from 62 clones. In TK, 21 RFLP types were detected from 58 clones. The genotypic diversity among the three sampling sites was 0·958, 0·636 and 0·821, respectively, indicating that the microbial diversity of SA was higher than at the other two stations. At SA, the most abundant RFLP type constituted 10% of all clones. The samples from SB and TK had dominant RFLP types which constituted 60% and 38% of the total clone libraries, respectively. The community structure of SA included many single-type clones, which were found only once in the clone libraries. This structure contrasted with that of the other two stations. Thirty-seven clones were selected and sequenced according to dendrograms derived from ARDRA, to cover most of the microbial diversity in the clone libraries. No clones were identical to any of the known 16S rRNA sequences or to each other. All sequences had >84·8% similarity to rDNA sequences retrieved from the DNA databases. Sequenced clones fell into five major lineages of the domain Bacteria: the gamma, delta and epsilon Proteobacteria, Gram-positive bacteria and the division Verrucomicrobia. At SA, the Verrucomicrobia and the three subclasses of the Proteobacteria were found. Most clone sequences belonged to the gamma Proteobacteria. The high-GC Gram-positive bacteria and the gamma subclass of the Proteobacteria were common at both SB and TK. Although the depths of SB and TK were very different, the community diversity inferred from ARDRA and the taxonomic position of the dominant clones were similar. All clones belonging to the high-GC Gram-positive bacteria collected from both SB and TK fell into the same cluster and are regarded as members of an unknown actinomycete group. The clone compositions were different at each sampling site, and clones of the gamma Proteobacteria and high-GC Gram-positive bacteria were dominant.
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- Genetics And Molecular Biology
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The genetic basis of the phase variation repertoire of lipopolysaccharide immunotypes in Neisseria meningitidis
The GenBank accession number for the sequence reported in this paper is U65788.
Neisseria meningitidis strains express a diverse range of lipopolysaccharide (LPS) structures that have been classified into 12 immunotypes. A feature of meningococcal LPS is the reversible, high-frequency switching of expression (phase variation) of terminal LPS structures. A number of studies are strongly suggestive of a key role for these terminal structures, and their phase-variable expression, in pathogenesis. In a previous study, a locus of three LPS biosynthetic genes, lgtABE, involved in the biosynthesis of one of these terminal structures, lacto-N-neotetraose, was described. The molecular mechanism of phase-variable expression of this structure is by high-frequency mutation in a homopolymeric tract of G residues in the lgtA gene. To investigate the genetic basis of the structural differences between the immunotypes, and the potential for strains to express alternative immunotypes, this locus was examined in all of the immunotype strains. Initially, the lgt locus of strain 126E, an L1 immunotype strain, was cloned and sequenced, revealing two active genes, lgtC and lgtE. The remnants of the lgtA and lgtB genes and an inactive lgtD gene were also present, indicating that the locus may have once contained five active genes, similar to a locus previously reported in Neisseria gonorrhoeae strain F62. Probes based on each of the lgt genes (ABCDE), and the recently reported lgtG gene, were used to determine the presence or absence of lgt genes within individual strains, allowing the prediction of the phase variation repertoire of these strains. Sequencing to determine the nature of homopolymeric tract regions within the lgt genes was carried out to establish the potential for LPS switching. In general, the set of strains examined could be sorted into two distinct groups: one group which phase-vary the α-chain extension via lgtA or lgtC but cannot make β-chain; the second group phase-vary the β-chain extension via lgtG but do not vary α-chain (lacto-N-neotetraose).
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The Candida albicans gene for mRNA 5′-cap methyltransferase: identification of additional residues essential for catalysis
The GenBank accession numbers for the nucleotide sequence of CaABD1 and hMet are AB020965 and AB020966, respectively.
The 5′-cap structure of eukaryotic mRNA is methylated at the terminal guanosine by RNA (guanine-N 7 -)-methyltransferase (cap MTase). Saccharomyces cerevisiae ABD1 (ScABD1) and human hMet (also called CMT1) genes are responsible for this enzyme. The ABD1 homologue was cloned from the pathogenic fungus Candida albicans and named C. albicans ABD1 (CaABD1). When expressed as a fusion with glutathione S-transferase (GST), CaAbd1p displayed cap MTase activity in vitro and rescued S. cerevisiae abd1Δ null mutants, indicating that CaABD1 specifies an active cap MTase. Although the human cap MTase binds to the human capping enzyme (Hce1p), which possesses both mRNA guanylyltransferase (mRNA GTase) and mRNA 5′-triphosphatase (mRNA TPase) activities, yeast two-hybrid analysis demonstrated that in yeast neither mRNA GTase nor mRNA TPase physically interacted with the Abd1 protein. Comparison of the amino acid sequences of known and putative cap MTases revealed a highly conserved amino acid sequence motif, Phe/Val-Leu-Asp/Glu-Leu/Met-Xaa-Cys-Gly-Lys-Gly-Gly-Asp-Leu-Xaa-Lys, which encompasses the sequence motif characteristic of divergent methyltransferases. Mutations in CaAbd1p of leucine at the second and the twelfth positions (so far uncharacterized) to alanine severely diminished the enzyme activity and the functionality in vivo, whereas those of leucine at the fourth, cysteine at the sixth, lysine at the eighth, and glycine at the tenth positions did not. Furthermore, valine substitution for the twelfth, but not for the second, leucine in that motif abolished the activity and functionality of CaAbd1p. Thus, it appears that leucine at the second and the twelfth positions in the motif, together with a previously identified acidic residue in the third, glycine at the sixth and glutamic acid at the eleventh positions, play important roles in the catalysis, and that side chain length is crucial for the activity at the twelfth position in the motif.
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The rpoS-dependent starvation-stress response locus stiA encodes a nitrate reductase (narZYWV) required for carbon-starvation-inducible thermotolerance and acid tolerance in Salmonella typhimurium
The starvation-stress response (SSR) of Salmonella typhimurium includes gene products necessary for starvation avoidance, starvation survival and virulence for this bacterium. Numerous genetic loci induced during carbon-source starvation and required for the long-term-starvation survival of this bacterium have been identified. The SSR not only protects the cell against the adverse effects of long-term starvation but also provides cross-resistance to other environmental stresses, e.g. thermal challenge (55 °C) or acid-pH challenge (pH 2·8). One carbon-starvation-inducible lac fusion, designated stiA was previously reported to be a σS-dependent SSR locus that is phosphate-starvation, nitrogen-starvation and H2O2 inducible, positively regulated by (p)ppGpp in a relA-dependent manner, and negatively regulated by cAMP:cAMP receptor protein complex and OxyR. We have discovered through sequence analysis and subsequent biochemical analysis that the stiA::lac fusion, and a similarly regulated lac fusion designated sti-99, lie at separate sites within the first gene (narZ) of an operon encoding a cryptic nitrate reductase (narZYWV) of unknown physiological function. In this study, it was demonstrated that narZ was negatively regulated by the global regulator Fnr during anaerobiosis. Interestingly, narZ(YWV) was required for carbon-starvation-inducible thermotolerance and acid tolerance. In addition, narZ expression was induced ∼20-fold intracellularly in Madin-Darby canine kidney epithelial cells and ∼16-fold in intracellular salts medium, which is believed to mimic the intracellular milieu. Also, a narZ1 knock-out mutation increased the LD50 ∼10-fold for S. typhimurium SL1344 delivered orally in the mouse virulence model. Thus, the previously believed cryptic and constitutive narZYWV operon is in fact highly regulated by a complex network of environmental-stress signals and global regulatory functions, indicating a central role in the physiology of starved and stressed cells.
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Mutational analysis of the Paracoccus denitrificans c-type cytochrome biosynthetic genes ccmABCDG: disruption of ccmC has distinct effects suggesting a role for CcmC independent of CcmAB
More LessThe GenBank accession number for the sequence determined in this work is Z71971.
Each of the Paracoccus denitrificans genes in the c-type cytochrome biogenesis gene cluster ccmABCDG, plus the two flanking genes ORF117 and hisH, were individually disrupted by Ω insertion. Resultant phenotypes were restored to the wild-type by complementation from a set of plasmids. All of the ccm genes, but neither ORF117 nor hisH, were required for c-type cytochrome biogenesis; only ccmG was also implicated in the biosynthesis of cytochrome aa 3. Disruption of ccmC or ccmG resulted in failure to grow on rich media, but disruption of ccmA, ccmB or ccmD did not. The ccmC mutant, but not the ccmA, ccmB or ccmD mutants, also exhibited the increased sensitivity to growth inhibition by oxidized thiol compounds previously observed for the ccmG mutant. In contrast to the ccmG mutant, however, growth of the ccmC mutant on rich media could not be restored by DTT. Siderophore biosynthesis and/or secretion by P. denitrificans was also attenuated by disruption of ccmC and ccmG but not of ccmA, ccmB or ccmD. These results indicate that CcmC can function independently of CcmA, CcmB and CcmD despite other evidence that these gene products form an ATP-binding cassette (ABC)-type-transporter with the subunit composition (CcmA)2-CcmB-CcmC or (CcmA)2-CcmB-CcmC-CcmD, and also suggest a possible link between the functions of CcmC and CcmG.
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Genetic and functional analysis of genes required for the post-modification of the polyketide antibiotic TA of Myxococcus xanthus
More LessThe EMBL accession number for the sequence reported in this paper is AJ132503.
The antibiotic TA of Myxococcus xanthus is a complex macrocyclic polyketide, produced through successive condensations of acetate by a type I PKS (polyketide synthase) mechanism. The genes encoding TA biosynthesis are clustered on a 36 kb DNA fragment, which has been cloned and analysed. The chemical structure of TA and the mechanism by which it is synthesized indicate the need for several post-modification steps, which are introduced into the carbon chain of the polyketide to form the final bioactive molecule. These include the addition of several carbon atoms originating from acetate carbonyl, three C-methylations, O-methylation and a specific hydroxylation. This paper reports the analysis of five genes which are involved in the post-modification of TA. Their functional analysis, by specific gene disruption, suggests that they may be essential for the production of the active antibiotic. The characteristics and organization of the genes suggest that they may be involved in the addition of the carbon atoms which arise from acetate.
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Direct evidence for mRNA binding and post-transcriptional regulation by Escherichia coli aconitases
More LessEscherichia coli contains a stationary-phase aconitase (AcnA) that is induced by iron and oxidative stress, and a major but less stable aconitase (AcnB) synthesized during exponential growth. These enzymes were shown to resemble the bifunctional iron-regulatory proteins (IRP1)/cytoplasmic aconitases of vertebrates in having alternative mRNA-binding and catalytic activities. Affinity chromatography and gel retardation analysis showed that the AcnA and AcnB apo-proteins each interact with the 3′ untranslated regions (3′UTRs) of acnA and acnB mRNA at physiologically significant protein concentrations. AcnA and AcnB synthesis was enhanced in vitro by the apo-aconitases and this enhancement was abolished by 3′UTR deletion from the DNA templates, presumably by loss of acn-mRNA stabilization by bound apo-aconitase. In vivo studies showed that although total aconitase activity is lowered during oxidative stress, synthesis of the AcnA and AcnB proteins and the stabilities of acnA and acnB mRNAs both increase, suggesting that inactive aconitase mediates a post-transcriptional positive autoregulatory switch. Evidence for an iron–sulphur-cluster-dependent switch was inferred from the more than threefold higher mRNA-binding affinities of the apo-aconitases relative to the holo-enzymes. Thus by modulating translation via site-specific interactions between apo-enzyme and relevant transcripts, the aconitases provide a new and rapidly reacting component of the bacterial oxidative stress response.
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The hydrophobic heptad repeat in Region III of Escherichia coli transcription factor sigma 54 is essential for core RNA polymerase binding
Escherichia coli transcription factor sigma 54 contains motifs that resemble closely those used for RNA polymerase II in mammalian cells, including two hydrophobic heptad repeats, a very acidic region and a glutamine-rich region. Triple changes in hydrophobic or multiple changes in acidic residues in Region III are known to severely impair core-binding ability. To investigate whether all the changes in triple mutants are necessary for core binding, site-directed mutagenesis was performed to create single and double mutants in the leucine or isoleucine residues in the heptad repeat in Region III. Single mutants showed no discernible loss of function. Double mutants showed partial protection of the −12 promoter element of the glnAp2 promoter due to the partial loss of their ability to bind core RNA polymerase. These mutations were deleterious to the function of sigma 54, which retained only 30–40% of wild-type mRNA levels. However, double mutants retained nearly normal ability to form open complexes. Two triple mutants created during previous work lost most, if not all, of their ability to bind core RNA polymerase, to protect the −12 promoter element of the glnAp2 promoter and to open the transcription start site. The two triple mutants produced about 20% or less than 10% of the wild-type transcripts from the glnAp2 promoter. These results demonstrate that the hydrophobic heptad repeat in Region III is essential for core RNA polymerase binding. Progressive loss of hydrophobicity of the hydrophobic heptad repeat in Region III of sigma 54 resulted in a progressive loss of core-binding ability, leading to the loss of −12 promoter element recognition and mRNA production.
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Absence of RNase III alters the pathway by which RNAI, the antisense inhibitor of ColE1 replication, decays
More LessRNAI is a short RNA, 108 nt in length, which regulates the replication of the plasmid ColE1. RNAI turns over rapidly, enabling plasmid replication rate to respond quickly to changes in plasmid copy number. Because RNAI is produced in abundance, is easily extracted and turns over quickly, it has been used as a model for mRNA in studying RNA decay pathways. The enzymes polynucleotide phosphorylase, poly(A) polymerase and RNase E have been demonstrated to have roles in both messenger and RNAI decay; it is reported here that these enzymes can work independently of one another to facilitate RNAI decay. The roles in RNAI decay of two further enzymes which facilitate mRNA decay, the exonuclease RNase II and the endonuclease RNase III, are also examined. RNase II does not appear to accelerate RNAI decay but it is found that, in the absence of RNase III, polyadenylated RNAI, unprocessed by RNase E, accumulates. It is also shown that RNase III can cut RNAI near nt 82 or 98 in vitro. An RNAI fragment corresponding to the longer of these can be found in extracts of an rnc + pcnB strain (which produces RNase III) but not of an rnc pcnB strain, suggesting that RNAI may be a substrate for RNase III in vivo. A possible pathway for the early steps in RNAI decay which incorporates this information is suggested.
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A family 26 mannanase produced by Clostridium thermocellum as a component of the cellulosome contains a domain which is conserved in mannanases from anaerobic fungi
More LessThe GenBank/EMBL accession number for the sequence reported in this paper is AJ242666.
Cellulosomes prepared by the cellulose affinity digestion method from Clostridium thermocellum culture supernatant hydrolysed carob galactomannan during incubation at 60 °C and pH 6·5. A recombinant phage expressing mannanase activity was isolated from a library of C. thermocellum genomic DNA constructed in λZAPII. The cloned fragment of DNA containing a putative mannanase gene (manA) was sequenced, revealing an ORF of 1767 nt, encoding a protein (mannanase A; Man26A) of 589 aa with a molecular mass of 66816 Da. The putative catalytic domain (CD) of Man26A, identified by gene sectioning and sequence comparisons, displayed up to 32% identity with other mannanases belonging to family 26. Immediately downstream of the CD and separated from it by a short proline/threonine linker was a duplicated 24-residue dockerin motif, which is conserved in all C. thermocellum cellulosomal enzymes described thus far and mediates their attachment to the cellulosome-integrating protein (CipA). Man26A consisting of the CD alone (Man26A′) was hyperexpressed in Escherichia coli BL21(DE3) and purified. The truncated enzyme hydrolysed soluble and insoluble mannan, displaying a temperature optimum of 65 °C and a pH optimum of 6·5, but exhibited no activity against other plant cell wall polysaccharides. Antiserum raised against Man26A′ cross-reacted with a polypeptide with a molecular mass of 70000 Da that is part of the C. thermocellum cellulosome. A second variant of Man26A containing the N-terminal segment of 130 residues and the CD (Man26A′′) bound to ivory-nut mannan and weakly to soluble Carob galactomannan and insoluble cellulose. Man26A′ consisting of the CD alone did not bind to these polysaccharides. These results indicate that the N-terminal 130 residues of mature Man26A may constitute a weak mannan-binding domain. Sequence comparisons revealed a lack of identity between this region of Man26A and other polysaccharide-binding domains, but significant identity with a region conserved in the three family 26 mannanases from the anaerobic fungus Piromyces equi.
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Genetic and biochemical characterization of the α and β components of a propionyl-CoA carboxylase complex of Streptomyces coelicolor A3(2)
More LessThe GenBank accession numbers for the accA1, aacA2 and pccB sequences determined in this work are AF113603, AF113604 and AF113605, respectively.
Two genes, accA1 and accA2, with nearly identical nucleotide sequences were cloned from Streptomyces coelicolor A3(2). The deduced amino acid sequences of the product of these two genes showed high similarity to BcpA2 of Saccharopolyspora erythraea and other biotin-containing proteins from different organisms assumed to be the α subunit of a propionyl-CoA carboxylase. A gene, pccB, encoding the carboxyl transferase subunit of this enzyme complex was also characterized. Strains disrupted in accA1 did not show any change in acetyl- or propionyl-CoA carboxylase activity, whilst cell-free extracts of a pccB mutant strain contained a reduced level of propionyl-CoA carboxylase. No mutants in accA2 could be isolated, suggesting that the gene may be essential. Heterologous expression of accA1, accA2 and pccB in Escherichia coli and in vitro reconstitution of enzyme activity confirmed that PccB is the β subunit of a propionyl-CoA carboxylase and that either AccA1 or AccA2 could act as the α component of this enzyme complex. The fact that accA2 mutants appear to be inviable suggests that this gene encodes a biotinylated protein that might be shared with other carboxyl transferases essential for the growth of S. coelicolor.
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A novel member of the subtilisin-like protease family from Bacillus subtilis
More LessaprX is a 1326 bp gene of Bacillus subtilis strain 168 that encodes a serine protease, probably intracellular, characterized by significant similarity with subtilisins, thermitases and pyrolysins. Transcription analysis, performed by RT-PCR and primer extension, allowed the localization of the active promoter and showed that aprX is expressed in stationary phase. The pattern of expression of aprX and its dependence on various transition state regulatory genes (degU, degQ, hpr, abrB, sinR), monitored by lacZ transcriptional fusions, are distinctive from those of subtilisin and other degradative enzymes. aprX is not essential for either growth or sporulation.
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Sequence analysis of three Bacillus cereus loci carrying PlcR-regulated genes encoding degradative enzymes and enterotoxin
The EMBL accession numbers for the sequences reported in this paper are given in Table 1 T1 .
PlcR is a pleiotropic regulator of extracellular virulence factors in the opportunistic human pathogen Bacillus cereus and the entomopathogenic Bacillus thuringiensis, and is induced in cells entering stationary phase. Among the genes regulated by PlcR are: plcA, encoding phosphatidylinositol-specific phospholipase C (PI-PLC); plc, encoding phosphatidylcholine-preferring phospholipase C (PC-PLC); nhe, encoding the non-haemolytic enterotoxin; hbl, encoding haemolytic enterotoxin BL (HBL); and genes specifying a putativeS-layer like surface protein and a putative extracellular RNase. By analysing 37·1 kb of DNA sequence surrounding hbl, plcA and plcR, 28 ORFs were predicted. Three novel genes putatively regulated by PlcR and encoding a neutral protease (NprB), a subtilase family serine protease (Sfp) and a putative cell-wall hydrolase (Cwh) were identified. The corresponding sfp and cwh genes were located in the immediate upstream region of plcA and could both be regulated by a putative PlcR-binding site positioned between the inversely transcribed genes. Similarly, nprB was positioned directly upstream and transcribed in the opposite orientation to plcR. Genes surrounding plcA, plcR and hblCDAB that were lacking an upstream PlcR regulatory sequence did not appear to serve functions apparently related to PlcR and did not exhibit a conserved organization in Bacillus subtilis.
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Insertional inactivation of hblC encoding the L2 component of Bacillus cereus ATCC 14579 haemolysin BL strongly reduces enterotoxigenic activity, but not the haemolytic activity against human erythrocytes
More LessHaemolysin BL (HBL) is a Bacillus cereus toxin composed of a binding component, B, and two lytic components, L1 and L2. HBL is also the enterotoxin responsible for the diarrhoeal food poisoning syndrome caused by several strains of B. cereus. The three genes encoding the HBL components constitute an operon and are transcribed from a promoter 608 bp upstream of the hblC translational start site. The first gene of the hbl operon, hblC, in the B. cereus type strain, ATCC 14579, was inactivated in this study. Inactivation of hblC strongly reduced both the enterotoxigenic activity of B. cereus ATCC 14579 and the haemolytic activity against sheep erythrocytes, while maintaining full haemolytic activity against human erythrocytes.
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PepR1, a CcpA-like transcription regulator of Lactobacillus delbrueckii subsp. lactis
More LessThe PepR1 protein from Lactobacillus delbrueckii subsp. lactis DSM 7290 shares extensive homology with catabolite-control proteins from various Gram-positive bacteria. Expression of the subcloned pepR1 gene allowed for partial complementation of a ccpA defect in Staphylococcus xylosus. The influence of PepR1 on transcription of the prolidase gene pepQ, which is located adjacent to pepR1, was examined by use of lacZ reporter gene fusions in Escherichia coli. PepR1 stimulated transcription initiation at the pepQ promoter about twofold, and this effect required the integrity of a 14 bp palindromic cre-like sequence located 74 nt upstream of pepQ. In gel-mobility-shift assays, PepR1 specifically interacted with the pepQ promoter region and also with DNA fragments covering the promoters of the pepX, pepI and brnQ genes of Lb. delbrueckii subsp. lactis, which encode two additional peptidases and a branched-chain amino acid transporter, respectively. cre-like elements were identified in each of these DNA fragments. Catabolite control of PepQ was demonstrated in Lb. delbrueckii subsp. lactis. During growth with lactose the enzyme activity was twofold higher than in the presence of glucose, and corresponding differences were also detected in the level of pepQ transcription.
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Synthesis of lactococcin 972, a bacteriocin produced by Lactococcus lactis IPLA 972, depends on the expression of a plasmid-encoded bicistronic operon
More LessThe GenBank accession number for the sequence reported in this paper is AJ002203.
Synthesis of lactococcin 972 is plasmid-encoded. An operon composed of two genes that encode pre-bacteriocin and a putative immunity protein has been identified. The first gene encodes a 91-residue polypeptide that is exported via a sec-dependent system to give the mature 66-aa bacteriocin. The immunity protein is a 563-residue polypeptide with seven potential transmembrane domains. Two transcripts were observed from this region: one comprises the whole operon and is synthesized during the exponential phase of growth while the other, which corresponds just to the bacteriocin structural gene, presents a maximum in exponential cultures but is still present in late-stationary-phase cells.
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IS1626, a new IS900-related Mycobacterium avium insertion sequence
More LessThe GenBank accession number for the IS1626 sequence determined in this work is AF071067.
An insertion sequence designated IS1626 was isolated and characterized from a Mycobacterium avium clinical strain. IS1626 was detected by high-stringency hybridization with the pMB22/S12 probe from IS900 of Mycobacterium paratuberculosis. IS1626 is 1418 bp in size and has a G+C content of 65 mol%. It has neither terminal inverted repeats nor flanking direct repeats. Analysis of three IS1626 insertion sites in the M. avium strain and the corresponding potential insertion sites in two IS1626-free M. avium strains indicated a consensus sequence of CATGCN(4–5)TCCTN(2)G for IS1626 insertion. In the three clones examined, IS1626 has the same orientation with respect to this target site. IS1626 has two major ORFs. ORF1179 encodes a predicted protein of 393 amino acids. ORF930, on the complementary strand of ORF1179, encodes a protein of 310 amino acids. The Shine–Dalgarno sequence for ORF930 is partially located in the flanking region, similar to other IS900-related elements. Analysis of the comparable features of insertion sequences and their variable occurrence in related organisms is useful for studying the evolution of these elements and their hosts.
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Context-sensitive transposition of IS6110 in mycobacteria
More LessThe rational use of IS6110 fingerprinting for studies of the molecular epidemiology and evolution of Mycobacterium tuberculosis requires understanding of the dynamics of transposition. In laboratory model systems, it has been shown that transposition is context-sensitive, i.e. it is influenced by the nature of the site in which the insertion sequence is presented. Stimulation of transposition by activation of an adjacent promoter supports the hypothesis that transposition occurs more readily from transcriptionally active locations. In addition, it has been shown that transposition can be enhanced by the expression of the transposase in trans. These findings imply that the frequency of transposition will vary substantially between different strains of M. tuberculosis, and furthermore that a hitherto stable strain may develop more rapid variation due to transposition into an active site. The use of IS6110 fingerprinting for the analysis of longer-range relationships between M. tuberculosis isolates therefore needs to be interpreted with care.
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- Pathogenicity And Medical Microbiology
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Phase variation of lic1A, lic2A and lic3A in colonization of the nasopharynx, bloodstream and cerebrospinal fluid by Haemophilus influenzae type b
More LessThe role of phase variation of lic1A, lic2A and lic3A in the ability of Haemophilus influenzae type b to colonize the nasopharynx, bloodstream and cerebrospinal fluid (CSF) of infants was investigated. This was achieved by using PCR to determine the number of 5′-CAAT-3′ repeats present in each gene, which is indicative of whether each ORF can be expressed. Multiple PCR products of different intensities were amplified from all three genes at each site sampled. This indicated that the nasopharynx, bloodstream and CSF were colonized by a heterogeneous population of organisms, expressing different combinations of lic genes. At each site however, a predominant PCR product was amplified from each gene, indicating that organisms with this genotype were the most abundant. The number of 5′-CAAT-3′ repeats in this predominant product varied depending upon whether organisms were isolated from the nasopharynx, bloodstream or CSF. These observations suggest that the expression of different combinations of lic genes may influence the efficiency with which H. influenzae colonizes the nasopharynx, bloodstream and CSF of infant rats.
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Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis
More LessThe EMBL accession number for the sequence in this paper is X96711.
Mycobacterium tuberculosis has innate resistance to a range of broad-spectrum antimicrobial agents. This may in part reflect the relative impermeability of the mycobacterial cell wall, but additional specific mechanisms may also be important. In the case of fosfomycin, it has been suggested that a key difference in the active site of the M. tuberculosis MurA enzyme might confer resistance. In Escherichia coli, fosfomycin covalently binds to a cysteine normally involved in the enzymic activity, while protein alignments predict an aspartate at this position in the M. tuberculosis MurA. In the present study, it is demonstrated that the wild-type M. tuberculosis MurA is indeed resistant to fosfomycin, and that it becomes sensitive following replacement of the aspartate residue in position 117 by a cysteine. In addition, the study illustrates the use of an inducible expression system in mycobacteria to allow functional characterization of an M. tuberculosis enzyme that is unstable during constitutive expression.
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Population biology of Streptococcus pneumoniae isolated from oropharyngeal carriage and invasive disease
The GenBank accession numbers for the sequences of the trpA/B alleles determined in this study are AF157817 to AF157826.
The population structure of Streptococcus pneumoniae in a sample of 134 carried antibiotic-susceptible isolates, and 53 resistant and susceptible invasive isolates, was examined using a DNA-based version of multilocus enzyme electrophoresis: multilocus restriction typing (MLRT). This involved RFLP analysis of PCR products generated from nine loci of housekeeping genes located around the pneumococcal chromosome. The combination of alleles at each of the nine loci gave an allelic profile or restriction type (RT). All carried (throat or nasopharyngeal) isolates from children or adults in Oxford and Manchester, UK, and from an HIV-seropositive cohort in Nairobi, Kenya, showed an epidemic population structure. Twelve carried clonal groups, each with different serotypes, were identified at both locations within the UK. Almost all of the carried clones examined (16/17) were found to possess identical RTs or sequence types (STs) to invasive isolates, indicating that frequently carried clones are also associated with cases of invasive disease. As expected from previous studies, the population of 53 invasive, mainly penicillin-resistant, isolates was also found to be at linkage equilibrium. Serotype switching was identified among 14% of RTs that possessed two or more members, or 5·7% of individual isolates within these RTs. In support of a population structure in which there is frequent recombination, there is also clear evidence that the trpA/B locus within pneumococci has evolved by horizontal gene transfer. A non-serotypable isolate from an HIV-seropositive patient in Kenya was clearly genetically distinct from other strains studied, with unique alleles at eight out of nine loci examined. However, it was initially identified as a pneumococcus by a 16S RNA gene probe (Gen-Probe), optochin susceptibility and the presence of pneumolysin and autolysin.
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Expression of cytotoxicity by potential pathogens in the standard Escherichia coli collection of reference (ECOR) strains
More LessThe GenBank accession numbers for the sequences reported in this paper are AF159702 and AF160993–161002.
The standard Escherichia coli collection of reference (ECOR) strains was examined for ability to exert cytotoxicity towards mammalian cells. A group of strains with functional haemolysin expression caused strong cytotoxicity and detachment in J774 macrophage cells as measured by lactate dehydrogenase release and as observed under a microscope. The expression of haemolysin was monitored by using antisera recognizing the E. coli α-haemolysin, the HlyA protein, and by quantitative haemolysis assays. The presence of the hlyA gene, which may be part of a pathogenicity island, was also confirmed. These analyses revealed that different ECOR strains express quantitatively different levels of haemolysin. One putative enteroaggregative E. coli (EAEC) strain was also found in the ECOR collection. The EAEC strain was characterized by the clump formation assay, PCR amplification of the EAEC DNA probe sequence and confirmative sequence analysis of the amplified fragment. The EAEC heat-stable enterotoxin 1 gene, astA, was found in 14% (10/72) of the ECOR strains and a consensus sequence for astA was proposed by comparing these sequences with those from pathogens. The astA gene appeared to be plasmid-located. Based on evidence from the work of other laboratories and from the present findings, it is concluded that the ECOR collection contains strains that may represent pathogenic E. coli. It is noted that caution is necessary when handling or disposing of those potentially pathogenic ECOR strains.
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- Physiology And Growth
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Analysis of the role of 7 kDa cold-shock proteins of Lactococcus lactis MG1363 in cryoprotection
Low-temperature adaptation and cryoprotection were studied in the lactic acid bacterium Lactococcus lactis MG1363. An approximately 100-fold increased survival after freezing was observed when cells were shocked to 10 °C for 4 h compared to mid-exponential-phase cells grown at 30 °C, indicating an active protection against freezing. Using two-dimensional gel electrophoresis a group of 7 kDa cold-induced proteins (CSPs) was identified that corresponds to a previously described family of csp genes of L. lactis MG1363 (Wouters et al., 1998 R32 , Microbiology 144, 2885–2893). The 7 kDa CSPs appeared to be the most strongly induced proteins upon cold shock to 10 °C. Northern blotting and two-dimensional gel electrophoresis showed that the csp genes were maximally expressed at 10 °C, while induction was lower at 20 and 4 °C. However, pre-incubation at 20 and 4 °C, as well as stationary-phase conditions, also induced cryoprotection (approx. 30-, 130- and 20-fold, respectively, compared to 30 °C mid-exponential phase). For all treatments leading to an increased freeze survival (exposure to 4, 10 and 20 °C and stationary-phase conditions), increased levels of three proteins (26, 43 and 45 kDa) were observed for which a role in cryoprotection might be suggested. Increased freeze survival coincides with increased CSP expression, except for stationary-phase conditions. However, the level of observed freeze protection does not directly correlate with the csp gene expression levels. In addition, for the first time specific overproduction of a CSP in relation to freeze survival was studied. This revealed that L. lactis cells overproducing CspD at 30 °C show a 2–10-fold increased survival after freezing compared to control cells. This indicates that the 7 kDa cold-shock protein CspD may enhance the survival capacity after freezing but that other factors supply additional cryoprotection.
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The Q15H mutation enables Crh, a Bacillus subtilis HPr-like protein, to carry out some regulatory HPr functions, but does not make it an effective phosphocarrier for sugar transport
Crh of Bacillus subtilis exhibits 45% sequence identity when compared to histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS). Crh can be phosphorylated by ATP at the regulatory Ser-46 and similar to P-Ser-HPr, P-Ser-Crh plays a role in carbon-catabolite repression. The sequence around the phosphorylatable Ser-46 in Crh exhibits strong similarity to the corresponding sequence of HPr of Gram-positive and a few Gram-negative bacteria. In contrast, the catalytic His-15, the site of PEP-dependent phosphorylation in HPr, is replaced with a glutamine in Crh. When Gln-15 was exchanged for a histidyl residue, in vitro PEP-dependent enzyme I-catalysed phosphorylation of the mutant Crh was observed. However, expression of the crhQ15H mutant allele did not restore growth of a ptsH deletion strain on the PTS sugars glucose, fructose or mannitol or on the non-PTS sugar glycerol. In contrast, Q15H mutant Crh could phosphorylate the transcriptional activator LevR as well as LevD, the enzyme IIA of the fructose-specific lev-PTS, which together with enzyme I, HPr and LevE forms the phosphorylation cascade regulating induction of the lev operon via LevR. As a consequence, the constitutive expression from the lev promoter observed in a ΔptsH strain became inducible with fructose when the crhQ15H allele was expressed in this strain.
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Glycerol transport and phosphoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of glycerol kinase in Thermus flavus
The genes glpK and glpF, encoding glycerol kinase and the glycerol facilitator of Thermus flavus, a member of the Thermus/Deinococcus group, have recently been identified. The protein encoded by glpK exhibited an unusually high degree of sequence identity (80·6%) when compared to the sequence of glycerol kinase from Bacillus subtilis and a similar high degree of sequence identity (64·8%) was observed when the sequences of the glycerol facilitators of the two organisms were compared. The work presented in this paper demonstrates that T. flavus is capable of taking up glycerol, that glpF and glpK are expressed constitutively and that glucose exerts a repressive effect on the expression of these genes. T. flavus was found to possess the general components of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) enzyme I and histidine-containing protein (HPr). These proteins catalyse the phosphorylation of T. flavus glycerol kinase, which contains a histidyl residue equivalent to His-232, the site of PEP-dependent, PTS-catalysed phosphorylation in glycerol kinase of Enterococcus casseliflavus. Purified glycerol kinase from T. flavus could also be phosphorylated with enzyme I and HPr from B. subtilis. Similar to enterococcal glycerol kinases, phosphorylated T. flavus glycerol kinase exhibited an electrophoretic mobility on denaturing and non-denaturing polyacrylamide gels that is different from the electrophoretic mobility of non-phosphorylated glycerol kinase. However, in contrast to PEP-dependent phosphorylation of enterococcal glycerol kinases, which stimulated glycerol kinase activity about 10-fold, phosphorylation of T. flavus glycerol kinase caused only a slight increase in enzyme activity.
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Impairment of sterol biosynthesis leads to phosphorus and calcium accumulation in Leishmania acidocalcisomes
The induction of the formation of inclusion vesicles in Leishmania amazonensis by the sterol biosynthesis inhibitors (SBI) ketoconazole and terbinafine has been reported previously. These compartments were recently identified as acidocalcisomes. By the use of electron spectroscopic imaging and energy loss spectroscopy, the presence of calcium, phosphorus and oxygen in the electron-dense inclusions located within the acidocalcisomes has been demonstrated. Endoplasmic reticulum cisternae formed membrane whorls which enclosed large portions of the cytoplasm and sometimes circumscribed acidocalcisomes. In addition, acid phosphatase activity, as well as the endocytic tracers horseradish peroxidase and gold-labelled transferrin and cystatin C were detected within these organelles in both SBI-treated and untreated parasites. These data suggest that impairment of sterol biosynthesis induces the biogenesis of acidocalcisomes and triggers an autophagic process that leads to intersection of the endosomal/lysosomal system with the acidocalcisomes.
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The yeast endosomal Na+/H+ exchanger, Nhx1, confers osmotolerance following acute hypertonic shock
More LessOsmotolerance in yeast is regulated by at least two distinct mechanisms. The acquired response occurs following long-term exposure to hypertonic medium and requires the induction of the HOG-MAP (high-osmolarity glycerol mitogen-activated protein) kinase cascade to increase levels of the osmolyte glycerol. The acute response occurs following sudden exposure to high osmotica and appears to be dependent on normal vacuole function. In this study it is reported that the yeast endosomal/prevacuolar Na+/H+ exchanger Nhx1 contributes to osmotolerance following sudden exposure to hyperosmotic media. Vacuolar shrinkage and recovery in response to osmotic shock was altered in the Δnhx1 null mutant. Our results also show that the osmotolerance conferred by Nhx1 contributes to the postdiauxic/stationary-phase resistance to osmotic stress and allows for the continued growth of cells until the acquired osmotolerance response can occur.
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Catalase activity is necessary for heat-shock recovery in Aspergillus nidulans germlings
To understand the molecular mechanisms induced by stress that contribute to the development of tolerance in eukaryotic cells, the filamentous fungus Aspergillus nidulans has been chosen as a model system. Here, the response of A. nidulans germlings to heat shock is reported. The heat treatment dramatically increased the concentration of trehalose and induced the accumulation of mannitol and mRNA from the catalase gene catA. Both mannitol and catalase function to protect cells from different reactive oxygen species. Treatment with hydrogen peroxide increased A. nidulans germling viability after heat shock whilst mutants deficient in catalase were more sensitive to a 50 °C heat exposure. It is concluded that the defence against the lethal effects of heat exposure can be correlated with the activity of the defence system against oxidative stress.
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- Corrigendum
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