- Volume 145, Issue 5, 1999
Volume 145, Issue 5, 1999
- Sgm Special Lecture
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- Microbiology Comment
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- Biochemistry
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Real-time monitoring of Bacillus subtilis endospore components by attenuated total reflection Fourier-transform infrared spectroscopy during germination
More LessChemical changes of particular Bacillus subtilis spore components were monitored by attenuated total reflection Fourier-transform infrared spectroscopy (ATR/FTIR) during spore germination on a ZnSe internal reflection element. Within minutes of the initiation of spore germination, significant changes in the amount of calcium dipicolinate (DPA-Ca) and proteins were noted in the wild-type strain. The changes in a germination mutant (strain 1G9, gerD) were similar to those in the wild-type strain, but the rates of change were slower. The changes in another germination mutant (strain 1G7, gerA) were very different from those in the first two strains: germination was slow and incomplete, and proteins and DPA-Ca remained unaltered throughout the course of the germination study. This technique thus offers a sensitive and non-destructive method for real-time monitoring of various cellular components during spore germination.
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Assay for UDPglucose 6-dehydrogenase in phosphate-starved cells: gene tuaD of Bacillus subtilis 168 encodes the UDPglucose 6-dehydrogenase involved in teichuronic acid synthesis
More LessA novel assay permitting the detection of UDPglucose 6-dehydrogenase activity in cell-free extracts obtained from phosphate-starved cultures of Bacillus subtilis is described. The critical step, the separation of phosphate-starvation-induced exo-enzymes, phosphatases and phosphodiesterases from the cytoplasmic fraction containing the UDPglucose dehydrogenase, was achieved by protoplasting and removal of the periplasmic fraction by protoplast washing. Using this method, the following were unambiguously demonstrated: (i) the presence in the cytoplasm of an enzymic activity oxidizing UDPglucose to UDPglucuronic acid, and (ii) that detection of the activity in whole-cell-free extracts is prevented by the presence of ‘periplasmic' enzymes catalysing the degradation of the sugar nucleotides. With this method, several B. subtilis 168 mutants unable to synthesize teichuronic acid were examined. Strains inactivated in gene tuaD, whose product shares homology with UDPglucose 6-dehydrogenase and GDPmannose 6-dehydrogenase from other organisms, were shown to lack UDPglucose 6-dehydrogenase activity. Anion exchange chromatography revealed that mutants deficient in tuaD lacked a cytoplasmic UDPglucuronate pool.
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The small GTP-binding protein Rho is expressed differentially during spore germination of Phycomyces blakesleeanus
Evidence has been obtained for the presence of the small 22 kDa GTP-binding Rho protein in dormant spores of Phycomyces blakesleeanus. Immunoblotting with a polyclonal antibody against RhoA revealed a soluble and membrane-associated 22 kDa protein. When [32P]ADP-ribosylated by Clostridium botulinum C3 exotoxin the protein had a pl of 5·7, a value similar to that reported for other RhoA proteins. The 22 kDa protein was expressed at all stages of growth investigated, but radiolabelling of the [32P]ADP-ribosylated protein increased when tube-formation occurred and decreased as the hyphae branched. Localization of RhoA during spore germination studied by immunofluorescence microscopy revealed the presence of RhoA in the cell membrane of the spore. When the spore started to swell, RhoA was observed as patches in the cell membrane which become concentrated in the neck region of the site of the protuberation tube, but this protein was never observed at the point of growth of the hyphal tip. The above results suggest that RhoA associated with one or more membrane proteins could participate in the molecular mechanism involved in maintaining cell integrity during the extrusion of the germ-tube of P. blakesleeanus.
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Yersiniabactin from Yersinia pestis: biochemical characterization of the siderophore and its role in iron transport and regulation
More LessA siderophore-dependent iron transport system of the pathogenic yersiniae plays a role in the pathogenesis of these organisms. The structure of the yersiniabactin (Ybt) siderophore produced by Yersinia enterocolitica has been elucidated. This paper reports the purification of Ybt from Yersinia pestis and demonstrates that it has the same structure as Ybt from Y. enterocolitica. Purified Ybt had a formation constant for Fe3+ of ~ 4×10-36. Addition of purified Ybt from Y. pestis enhanced iron uptake by a siderophore-negative (irp2)strain of Y. pestis. Maximal expression of the Ybt outer-membrane receptor, Psn, in this strain was dependent upon exogenously supplied Ybt. Regulation of Psn expression by Ybt occurred at the transcriptional level. Y. pestis DNA was used to construct irp2 and psn mutations in Yersinia pseudotuberculosis. The irp2 mutant strain no longer synthesized Ybt and the psn mutant strain could not use exogenously supplied Ybt. As in Y. pestis, Ybt was required for maximal expression of Psn. Regulation by Ybt occurred at the transcriptional level. In contrast to Y. pestis, in which a psn mutation does not repress synthesis of Ybt siderophore or expression of the iron-regulated HMWP1 and HMWP2 proteins, the same mutation in Y. pseudotuberculosis partially repressed these products.
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The NucE and NucD lysis proteins are not essential for secretion of the Serratia marcescens extracellular nuclease
More LessThe nuclease of Serratia marcescens is an extracellular protein encoded by the nucA gene. Pre-nuclease carries a typical 21-amino-acid N-terminal signal sequence that interacts with the Sec machinery to allow the translocation of nuclease to the periplasm. In Escherichia coli the nuclease remains in the periplasm; however, S. marcescens has the capacity to secrete nuclease extracellularly. The nucC operon carrying the nucEDC genes of S. marcescens has been identified previously. NucC is a transcriptional activator necessary for expression of nuclease as well as the extracellular bacteriocin 28b. NucE resembles and can act as a bacteriophage holin, whereas NucD has homology to bacteriophage lysozyme-like proteins. When present on a multicopy plasmid, the nucC operon, and specifically the nucED genes, appeared to allow extracellular secretion of nuclease from E. coli. Here experiments are reported which demonstrate that, when the nucC operon was placed in the E. coli chromosome in single copy, nuclease secretion was lost and nuclease remained periplasmic. The converse experiment, deletion of the nucE and nucD genes from the chromosome of S. marcescens, likewise had no effect on nuclease secretion by S. marcescens. It is concluded therefore that NucD and NucE are not necessary for nuclease secretion.
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- Development And Structure
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Structural analysis of Bacillus megaterium KM spore peptidoglycan and its dynamics during germination
More LessThe composition and structure of peptidoglycan from dormant spores of Bacillus megaterium KM and its dynamics during germination were investigated. Amino acid analysis and mass spectrometry identified 21 muropeptides resolved by reverse phase HPLC following digestion of peptidoglycan with Cellosyl. The basic structure of peptidoglycan in B. megaterium spores is similar to that of Bacillus subtilis: 44·2% of muramic acid residues are substituted with δ-lactam, 28·8% with single L-alanine, 25·1% with tetrapeptide and only 1·8% with tripeptide. The cross-linking index of the spore peptidoglycan, determined from muropeptides resolved by reverse phase HPLC, was 2·2% per muramic acid. Spore peptidoglycan contains 2·9% of muropeptides with unsubstituted N-acetylmuramic acid. These muropeptides are likely to be intermediate products of δ-lactam formation. Analysis of muropeptide dynamics during germination revealed the activity of at least two hydrolytic enzymes, an N-acetylglucosaminidase and a lytic transglycosylase. A 4 M LiCl extract from 30 min germinated spores of B. megaterium KM caused ‘germination-like' changes to permeabilized spores of B. megaterium and B. subtilis but not those of a B. subtilis cwID mutant. Muropeptide analysis of the treated spores revealed the presence of products generated by the activity of a glucosaminidase.
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Inactivation of the KlPMR1 gene of Kluyveromyces lactis results in defective cell-wall morphogenesis
More LessThe P-type Ca2+-ATPases are the transporters responsible for calcium homeostasis in the cell compartments of eukaryotes. The KlPMR1 gene of Kluyveromyces lactis encodes a P-type Ca2+-ATPase, which is functionally and structurally homologous to Pmr1p of Saccharomyces cerevisiae, the calcium pump localized in the Golgi membranes. In this work, a novel involvement of KlPmr1p in cell-wall morphogenesis of K. lactis is reported. Klpmr1 Δ cells exhibited the loss of outer-chain extension in the glycosylation of secreted proteins. The absence of KlPmr1p resulted in the accumulation of round, large cells with an abnormally thick cell wall, as revealed by transmission electron microscopy. The deletant strain also showed a delocalized deposition of chitin in the lateral cell wall accompanied by an unbalanced ratio of insoluble to soluble glucans. These morphological defects were accompanied by the presence of irregularly shaped nuclei and by a DNA content greater than 2n. Addition of 10 mM Ca2+ to the medium of the Klpmr1 Δ strain reversed the chitin-deposition impairment, recovered the alteration to the glucan ratio and restored a normal thickness of the cell wall. The mutant cells resumed wild-type size, shape and nuclear morphology but the DNA content indicated the persistence of defects in the co-ordination between DNA replication and cell division. The glycosylation defects were completely unaffected by the calcium supplement. These results indicate that calcium homeostasis controlled by KlPmr1p plays an important role in the cell-wall morphogenesis of K. lactis.
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Characterization of glycolipids from Meiothermus spp.
More LessThin-layer chromatographic analysis of the polar lipids of Meiothermus strains revealed two glycolipid bands with similar chromatographic mobility to the major glycolipid of Thermus strains. In this study the glycolipids from the type strains of Meiothermus ruber, Meiothermus chliarophilus, Meiothermus silvanus and Meiothermus cerbereus were characterized using GC, GC/MS, fast atom bombardment MS and chemical methods. All strains contained dihexosyl-(N-acyl)hexosaminylglucosyl diacylglycerols, related in structure to the major glycolipid of Thermus strains but varying in their fatty acylation pattern. The detection of two glycolipid bands by TLC in Meiothermus spp. was attributable to the invariable presence of 2-hydroxyacyl groups N-linked to the hexosamine of the polar head group which cause the glycolipids to be more strongly retained on silica TLC plates than 3-hydroxy or non-hydroxylated N-acyl glycolipids of similar structure that are also present. M. silvanus contained, in addition to these glyceroglycolipids, several glycolipids which were linked to acylated branched octadecanediols rather than to glycerol. The presence of glycolipids containing 2-hydroxyacyl groups N-linked to hexosamine appears to be a stable phenotypic marker that distinguishes the genus Meiothermus from the genus Thermus.
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- Genetics And Molecular Biology
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Two genes from Bacillus subtilis under the sole control of the general stress transcription factor σB
More LessThe general stress response of Bacillus subtilis is triggered by a variety of environmental and metabolic stresses which activate the σB transcription factor. Among the more than 100 genes controlled by σB (the csb genes), the functions identified thus far include resistance to oxidative stress, resistance to protein denaturation and resistance to osmotic stress. To understand the breadth of functions in which csb genes participate, the transcriptional organization and predicted products of two such genes previously identified in a screen for σB-dependent lacZ fusions were analysed. The csb-22::Tn917lacZ and csb-34::Tn917lacZ fusions are unusual among csb genes in that their expression appears to be completely dependent upon σB. By plasmid-integration experiments, fusion analyses and site-directed mutagenesis, stress-inducible, σB-dependent promoters for both these fusions were identified. The csb-34 fusion marked an ORF (yxcC or csbC) which by sequence analysis lay in a monocistronic transcriptional unit. This ORF encoded a predicted 461-residue product which had high identity with Class I sugar transporters of the major facilitator superfamily. It was speculated that the csbC product could serve either a nutritional or an osmotic protection function. In contrast, the csb-22 fusion identified an ORF (ywmG or csbD) which appeared to be the second gene of a two-gene operon. This ORF encoded a predicted 62-residue product which resembled a small Escherichia coli protein of unknown function. The σB-dependent promoter lay immediately upstream from csbD and appeared to be an internal promoter for the operon.
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Different temporal and spatial expression of two hydrophobin-encoding genes of the edible mushroom Agaricus bisporus
In a search for genes that are only expressed in fruit bodies of the basidiomycete Agaricus bisporus, two cDNAs, hypA and hypB that encode hydrophobins have been isolated previously. In this study, the structure of hypB is resolved and it is shown that the two genes are differentially expressed, indicating that the encoded hydrophobins serve different functions in A. bisporus mushrooms. hypB encodes a polypeptide (HYPB) of 119 aa that shows little sequence identity with HYPA apart from the characteristic arrangement of eight cysteines found exclusively in hydrophobins. The temporal and spatial expression of the two hydrophobin-encoding genes during fruit body development was compared using Northern analysis and in situ hybridization. Accumulation of hypA mRNA was found in tissue fractions consisting of undifferentiated white hyphae. In situ hybridization showed that the highest hypA mRNA levels are not found in the outermost cell layers of the pileipellis but in the cell layers adjacent to that. The highest level of expression of hypB occurs early in development when the primordium differentiates into densely packed, randomly oriented cap hyphae and loosely packed, vertically oriented stipe hyphae. In mature mushrooms, a strong accumulation of hypB transcripts was found only in the transitional zone between cap and stipe tissue, demonstrating that transcription regulation of hypB is clearly distinct from hypA.
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Changes in Aspergillus nidulans gene expression induced by bafilomycin, a Streptomyces-produced antibiotic
More LessIn natural environments bacteria and filamentous fungi often compete for the same resources. Consequently, production of antibiotic secondary metabolites and defence mechanisms against these compounds have evolved in these organisms. An experimental model has been developed to study the response in fungi exposed to one such antibiotic. The filamentous fungus Aspergillus nidulans was treated with bafilomycin B1, a Streptomyces-produced antibiotic which reduces radial growth rate and induces morphological changes in fungi. mRNA differential display was used to study changes in fungal gene expression. For five genes, changes in abundance of the corresponding mRNAs, directly or indirectly caused by bafilomycin, were observed. Of these, three were up-regulated and two repressed. With four of these the change in mRNA abundance measured ranged from 10- to 60-fold. However, for one gene the mRNA was only detected after bafilomycin treatment. One of the down-regulated mRNAs encodes ASPND1, a glycoprotein that belongs to a known family of antigens identified in aspergilloma patients. One up-regulated mRNA shows sequence similarities, at the amino acid level, with a cell-wall protein of Saccharomyces cerevisiae. The remaining three genes were also cloned and sequenced; their sequences do not correspond to known genes in A. nidulans, and no similarities with published nucleotide or protein sequences in other organisms were found. These results indicate the feasibility of using mRNA differential display to study interactions between bacteria and filamentous fungi.
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Purification of geranylgeranyltransferase I from Candida albicans and cloning of the CaRAM2 and CaCDC43 genes encoding its subunits
All previously characterized protein geranylgeranyltransferases I (GGTase I) are heterodimeric zinc metalloenzymes which catalyse geranylgeranylation of a cysteine residue in proteins containing a C-terminal CaaL motif (C, Cys; a, aliphatic amino acid; L, Leu). The α and β subunits of GGTase I of Saccharomyces cerevisiae are encoded by RAM2 and CDC43, respectively, and are essential for yeast viability. The authors are therefore investigating the role of geranylgeranylation in the related pathogenic yeast, Candida albicans, which is the most prevalent human fungal pathogen. GGTase I was purified to near homogeneity and also found to be a heterodimeric magnesium-dependent, zinc metalloenzyme displaying selectivity for CaaL-containing protein substrates. GGTase I peptide sequences were obtained from the purified protein and used to clone the genes encoding both subunits. CaRAM2 and CaCDC43 encode proteins that are 42 and 34% identical to their corresponding S. cerevisiae homologues, respectively, and 30% identical to their human homologues. Despite the limited overall homology, key zinc- and substrate-binding residues of the β subunit (Cdc43p) are conserved. A unique feature of CaCdc43p is a tract of polyasparagine whose length varies from 6 to 17 residues among C. albicans strains and between alleles. Coexpression of both CaCDC43 and CaRAM2 under their native promoters complemented the ts defect of a S. cerevisiae cdc43 mutant but expression of the β-subunit alone did not correct the growth defect, suggesting that hybrid GGTase I heterodimers are nonfunctional.
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Involvement of outer-membrane proteins in the aggregation of Azospirillum brasilense
More LessA bioassay was developed to investigate biological factors involved in the aggregation of Azospirillum brasilense strain Cd. Cells were grown for 24 h under aggregation-inducing and non-aggregation-inducing conditions (high and low C:N, respectively) and sonicated for 20 s. The cells were washed by centrifugation and resuspended in potassium phosphate buffer containing the two types of sonication extract. A greater extent of aggregation and higher flocculation were observed after 2–3 h incubation in the presence of sonicates from cells grown at high C:N (H-cells) compared to cells grown at low C:N. Flocculation did not occur after incubation of these cells in phosphate buffer. Boiled or proteinase K-treated sonicates originating from H-cells had lower aggregation-inducing capacity. After fractionation of the crude sonicate, both the outer-membrane protein (OMP) and the total membrane (mostly OMP) fractions possessed relatively high aggregation specific activities. The aggregation-inducing capacity of the OMP fraction strongly correlated with its protein concentration in the bioassay. Treatment of this fraction with proteinase K also decreased its aggregation-inducing activity. These findings suggest that OMPs are involved in the aggregation process of cells of A. brasilense.
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Transposition of IS117 of Streptomyces coelicolor A3(2) in Mycobacterium smegmatis
More LessDerivatives of IS117, the Streptomyces coelicolor A3(2) 2·6 kb minicircle, transpose efficiently in Mycobacterium smegmatis, targeting chromosomal sites resembling translation start signals. Two IS117 derivatives, plJ4696 and plJ4697, containing a Streptomyces hygromycin-resistance gene in opposite orientations were introduced into M. smegmatis by electroporation and found to integrate into one of three specific sites. Integrations at sites A and B were frequent while integration at site C was observed only once. Only one site was occupied in each transformant. Sites A and B had either single or tandem integrations. PFGE analysis located these sites on different genomic Asel fragments. The sequences of the chromosome-IS117 junctions confirmed that integration was via the same IS117 attachment site as in Streptomyces, that there was no target site duplication, and that the orientation of IS117 at each site was fixed. In contrast to the situation in Streptomyces lividans, no deletions were created by the transposition and no circular forms could be detected. Comparison of the three M. smegmatis chromosomal IS117 target sites (att B) with known primary and secondary S. lividans att B sites showed that only a 2 bp ‘AG’ sequence at the crossover point was conserved. Dividing the att B sites into two groups produced two longer consensus target sites, GtcAAGg and gCCGATAGg. Most of the IS117 target sites resemble translational start sites, and site C resembles strongly the amino-terminal sequence of a Mycobacterium tuberculosis aminopeptidase. The level of hygromycin resistance in the transformants was high and independent of the site of integration, the number of copies integrated, or the orientation of the hyg gene. plJ4696 at all three sites was stable in M. smegmatis in the absence of selection for at least 60 cell divisions. plJ4696, plJ4697 and other IS117 derivatives are promising vectors for the stable, integrative cloning of genes in M. smegmatis.
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gdhB, a gene encoding a second quinoprotein glucose dehydrogenase in Pantoea citrea, is required for pink disease of pineapple
More LessThe pink disease of pineapple, caused by the bacterium Pantoea citrea, is characterized by a dark coloration on fruit slices after canning. A glucose dehydrogenase (Gdh) encoded by the gdhA gene has been implicated in the colour formation activity of P. citrea. In this paper it has been shown that P. citrea contains a second, homologous gdh gene and its product, GdhB, represents the main source of Gdh activity in this organism. Unlike gdhA, gdhB is constitutively expressed during the exponential phase of growth and is induced in stationary phase. A previously isolated chemical mutant, CMC6, which is deficient in Gdh activity and pink disease formation, failed to express gdhB during the stationary phase of growth. The CMC6 mutant can be complemented by a 54 bp DNA fragment located upstream of gdhA. This fragment, which contains an operator-like 11 bp inverted repeat, strongly enhances the expression of gdhA, probably by titrating away a negative effector of its expression. These results illustrate the complex interplay operating between the two gdh genes and emphasize the role of glucose metabolism in the pathway leading to pink disease.
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Effects of gene disruptions in the nisin gene cluster of Lactococcus lactis on nisin production and producer immunity
More LessThe lantibiotic nisin is produced by several strains of Lactococcus lactis subsp. lactis. The chromosomally located gene cluster nisABTCIPRKFEG is required for biosynthesis, development of immunity, and regulation of gene expression. Inframe deletions in the nisB and nisT genes, and disruption of nisC by plasmid integration, eliminated nisin production and resulted in a strongly reduced level of immunity of the strains. The transcription of two nisin operons was inactivated in these mutant strains, but could be restored by addition of small amounts of nisin to growing cultures. The immunity levels of the mutants were also raised by adding nisin to growing cultures, albeit not to wild-type level. A strain with an in-frame deletion in the nisl gene was still able to produce active nisin, but the production and immunity levels were markedly lower. By measuring immunity levels of the knock-out strains and determining mRNA levels, it is concluded that Nisl has an important function for nisin immunity and must cooperate with nisFEG-encoded proteins to provide a high level of immunity. Maximal immunity could not be obtained in the mutant strains, probably because the wild-type transcription levels from nisA and nisF promoters are not reached when essential nis genes are disrupted. Using Southern hybridization with a consensus promoter probe, no other DNA sequences similar to the nisA and nisF promoters could be detected, indicating that these two elements are probably the only ones in the chromosome regulated by nisin and are thus the only ones involved in the regulation of producer immunity.
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Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath
More LessGenes for the subunits of particulate methane monooxygenase, PmoABC, have been sequenced from the γ-proteobacterial methanotroph Methylococcus capsulatus Bath. M. capsulatus Bath contains two complete copies of pmoCAB, as well as a third copy of pmoC. The two pmoCAB regions were almost identical at the nucleotide sequence level, differing in only 13 positions in 3183 bp. At the amino acid level, each translated gene product contained only one differing residue in each copy. However, the pmoC3 sequence was more divergent from the two other pmoC copies at both the far N-terminus and far C-terminus. Chromosomal insertion mutations were generated in all seven genes. Null mutants could not be obtained for pmoC3, suggesting that it may play an essential role in growth on methane. Null mutants were obtained for pmoC1, pmoC2, pmoA1, pmoA2, pmoB1 and pmoB2. All of these mutants grew on methane, demonstrating that both gene copies were functional. Copy 1 mutants showed about two-thirds of the wild-type whole-cell methane oxidation rate, while copy 2 mutants showed only about one-third of the wild-type rate, indicating that both gene copies were necessary for wild-type particulate methane monooxygenase activity. It was not possible to obtain double null mutants that were defective in both pmo copies, which may indicate that some expression of pMMO is important for growth.
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Expression of the Oenococcus oeni trxA gene is induced by hydrogen peroxide and heat shock
More LessSequencing of the DNA region located upstream of the α-acetolactate synthase and decarboxylase (alsS-alsD) cluster of Oenococcus oeni allowed identification of an ORF, named trxA. This encodes a protein of 104 amino acids very similar to known thioredoxins. The protein encoded by the cloned fragment was able to complement Escherichia coli strains lacking a functional thioredoxin. Considering the results of protein sequence comparisons and complementation experiments, it was concluded that the trxA gene encodes a functional thioredoxin. Studies of trxA expression showed that the abundance of trxA mRNA was similar during all growth stages. A significant increase in trxA mRNA levels was observed in the presence of hydrogen peroxide in the medium or after heat shock. A single transcriptional start site was determined with total RNA isolated from cells subjected or not subjected to oxidative stress or heat shock. In each case the same promoter region was identified and shown to have a high similarity to the consensus promoter sequence of Gram-positive bacteria, as well as to that of E. coli and the previously mapped promoters from O. oeni.
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