- 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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Structural and putative regulatory genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii
More LessSix genes involved in cellulose synthesis in Rhizobium leguminosarum bv. trifolii were identified using Tn5 mutagenesis. Four of them displayed homology to the previously cloned and sequenced Agrobacterium tumefaciens cellulose genes celA, celB, celC and celE. These genes are organized similarly in R. leguminosarum bv. trifolii. In addition, there were strong indications that two tandemly located genes, celR1 and celR2, probably organized as one operon, are involved in the regulation of cellulose synthesis. The deduced amino acid sequences of these genes displayed a high degree of similarity to the Caulobacter crescentus DivK and PleD proteins that belong to the family of two-component response regulators. This is to our knowledge the first report of genes involved in the regulation of cellulose synthesis. Results from attachment assays and electron microscopic studies indicated that cellulose synthesis in R. leguminosarum bv. trifolii is induced upon close contact with plant roots during the attachment process.
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Functional analysis of the O antigen glucosylation gene cluster of Shigella flexneri bacteriophage SfX
More LessPrevious studies have shown that Shigella flexneri bacteriophage X (SfX) encodes a glucosyltransferase (GtrX, formerly Gtr), which is involved in O antigen modification (serotype Y to serotype X). However, GtrX alone can only mediate a partial conversion. More recently, a three-gene cluster has been identified next to the attachment site in the genome of two other S. flexneri bacteriophages (i.e. SfV and SfII). This gene cluster was postulated to be responsible for a full O antigen conversion. Here it is reported that besides the gtrX gene, the other two genes in the gtr locus of SfX were also involved in the O antigen modification process. The first gene in the cluster (gtrA) encodes a small highly hydrophobic protein which appears to be involved in the translocation of lipid-linked glucose across the cytoplasmic membrane. The second gene in the cluster (gtrB) encodes an enzyme catalysing the transfer of the glucose residue from UDP-glucose to a lipid carrier. The third gene (gtrX) encodes a bacteriophage-specific glucosyltransferase which is largely responsible for the final step, i.e. attaching the glucosyl molecules onto the correct sugar residue of the O antigen repeating unit. A three-step model for the glucosylation of bacterial O antigen has been proposed.
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Molecular analysis of the recA gene and SOS box of the purple non-sulfur bacterium Rhodopseudomonas palustris no. 7
More LessThe recA gene of the purple non-sulfur bacterium Rhodopseudomonas palustris no. 7 was isolated by a PCR-based method and sequenced. The complete nucleotide sequence consists of 1089 bp encoding a polypeptide of 363 amino acids which is most closely related to the RecA proteins from species of Rhizobiaceae and Rhodospirillaceae. A recA-deficient strain of R. palustris no. 7 was obtained by gene replacement. As expected, this strain exhibited increased sensitivity to DNA-damaging agents. Transcriptional fusions of the recA promoter region to lacZ confirmed that the R. palustris no. 7 recA gene is inducible by DNA damage. Primer extension analysis of recA mRNA located the recA gene transcriptional start. A sequential deletion of the fusion plasmid was used to delimit the promoter region of the recA gene. A gel mobility shift assay demonstrated that a DNA-protein complex is formed at this promoter region. This DNA-protein complex was not formed when protein extracts from cells treated with DNA-damaging agents were used, indicating that the binding protein is a repressor. Comparison of the minimal R. palustris no. 7 recA promoter region with the recA promoter sequences from other α-Proteobacteria revealed the presence of the conserved sequence GAACA-N6-G(A/T)AC. Site-directed mutations that changed this consensus sequence abolished the DNA-damage-mediated expression of the R. palustris recA gene, confirming that this sequence is the SOS box of R. palustris and probably plays the same role in other α-Proteobacteria.
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- Genomics
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Nucleotide sequence of the Bacillus subtilis temperate bacteriophage SPβc2
The Bacillus subtilis 168 chromosomal region extending from 184° to 195°, corresponding to prophage SPβ, has been completely sequenced using DNA of the thermoinducible SPβc2 mutant. This 134416 bp segment comprises 187 putative ORFs which, according to their orientation, were grouped into three clusters. Compared to its host, SPβc2 is characterized by a lower G+C content, shorter mean ORF length, as well as a different usage of start codons. Nearly 75% of predicted ORFs do not share significant homologies to sequences in available databases. The only highly similar proteins to SPβc2-encoded ones are host paralogues. SPβc2 promoter regions contain SOS box consensus sequences and a repeated motif, designated SPβ repeated element (SPBRE), that is absent from the host genome. Gene sspC, encoding the small acid-soluble protein C, that has been previously sequenced and mapped to the vicinity of the SPβ region, was found to be part of the prophage.
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- Pathogenicity And Medical Microbiology
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Proteolytic cleavage of the A subunit is essential for maximal cytotoxicity of Escherichia coli 0157:H7 Shiga-like toxin-1
More LessMembers of the bacterial Shiga toxin family consist of a single A subunit that is non-covalently associated with a pentamer of B subunits. These toxins bind to receptors on susceptible mammalian cells and enter the cells by endocytic uptake. During cell entry, the 32 kDa A subunit is cleaved by the membrane-anchored protease furin to generate a catalytically active, 27·5 kDa A1 fragment and a 4·5 kDa A2 fragment. Previous studies have shown that mutating the furin site to prevent cleavage did not significantly affect toxin potency, suggesting that cleavage is not required for toxin activity. Here it is confirmed that preventing cleavage at the usual processing site does not prevent proteolytic processing of the Escherichia coli Shiga-like toxin-1 A subunit. However, simultaneous mutation of both the primary furin-recognition site and a nearby putative furin cleavage site did prevent intracellular processing of the A subunit. Comparison of the cytotoxicities of purified recombinant toxins to cultured mammalian cells demonstrated that even on prolonged incubation with toxin, the unprocessed mutant was 60-fold less toxic than the wild-type protein or other mutants still capable of being proteolytically processed during cell entry.
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The large plasmids of Shiga-toxin-producing Escherichia coli (STEC) are highly variable genetic elements
More LessShiga-toxin-producing Escherichia coli (STEC) of different serotypes are known to harbour large plasmids. The aim of this study was to investigate, using the example of the plasmid-encoded serine protease EspP, whether these plasmids are a uniform genetic element present in STEC. Examination of 201 diarrhoeagenic E. coli strains using a newly developed espP-specific PCR showed that espP is specific for STEC and present in 57% of STEC belonging to 16 different serotypes. The espP genes of the 16 STEC serotypes varied to a certain extent, as shown by nucleotide sequence and restriction enzyme analyses, but the DNA regions adjacent to the espP gene were completely different. When two further STEC-plasmid markers, the catalase-peroxidase gene katP and the enterohaemorrhagic E. coli-haemolysin gene EHEC-hlyA were included, many combinations of the three markers were found, depending in part on the serotype. In addition, strains possessing none of the three markers still harboured large plasmids. In the most prevalent STEC serogroup, O157, it was observed that the plasmid of sorbitol-fermenting STEC O157:H- lacks the espP and katP genes although both genes are present in the plasmid of the non-sorbitol-fermenting STEC O157:H7. The EHEC-hlyA gene, however, is present in both. In conclusion, this study shows that the large plasmids of STEC are not uniform genetic elements but heterogeneous in both their gene composition and arrangement.
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Fimbriae- and flagella-mediated association with and invasion of cultured epithelial cells by Salmonella enteritidis
More LessSalmonella enteritidis expresses flagella and several finely regulated fimbriae, including SEF14, SEF17 and SEF21 (type 1). A panel of mutants was prepared in three strains of S. enteritidis to elucidate the role of these surface appendages in the association with and invasion of cultured epithelial cells. In all assays, the naturally occurring regulatory-defective strain 27655R associated with tissue culture cells significantly more than wild-type progenitor strains LA5 and S1400/94. Compared with wild-type strains, SEF14 mutants had no effect on association and invasion, whereas SEF17, SEF21 and aflagellate mutants showed significant reductions in both processes. Histological examination suggested a role for SEF17 in localized, aggregative adherence, which could be specifically blocked by anti-SEF17 sera and purified SEF17 fimbriae. SEF21-mediated association was neutralized by mannose and a specific monoclonal antibody, although to observe enhanced association it was necessary for the bacteria to be in fimbriate phase prior to infection. Additionally, aflagellate mutants associated and invaded less than motile bacteria. This study demonstrated the potential for multifactorial association and invasion of epithelial cells which involved SEF17 and SEF21 fimbriae, and flagella-mediated motility.
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- Physiology And Growth
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Varying division planes of secondary constrictions in spheroidal Escherichia coli cells
More LessPlanes of successive divisions in Escherichia coli have been proposed to be either parallel or perpendicular to each other, restricted to one or two dimensions. To test the hypothesis that divisions can occur in planes alternating in three dimensions, a method was developed to generate cells with secondary constrictions during growth in suspension. The method involves a combination of thymine limitation (to manipulate chromosome replication rate) and mecillinam treatment (to inhibit penicillin-binding protein 2). The former modifies timing of terminations, the latter results in spheroidal cells. Such cells displayed secondary constrictions after adding deoxyguanosine (accelerating replication rate), thus temporarily enhancing division signals. The successive constrictions were seen to develop in planes that were tilted relative to each other, and in positions related to those of the nucleoids, visualized by staining with DAPI (4',6-diamidino-2-phenylindole dihydrochloride hydrate). Visualizing cell envelopes with FM 4–64 by confocal scanning laser microscopy supported the conclusion that planes of successive divisions can alternate in three dimensions.
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Anaerobic oxidations of cysteate: degradation via L-cysteate: 2-oxoglutarate aminotransferase in Paracoccus pantotrophus
More LessAnoxic, fresh-water enrichment cultures to oxidize different organosulfonates were set up with nitrate, ferric iron or sulfate as electron acceptors. Pure cultures were easily obtained with two naturally occurring sulfonates, cysteate (2-amino-3-sulfopropionate) and taurine (2-aminoethanesulfonate), under nitrate-reducing conditions. These two sulfonates were also oxidized during reduction of iron(III), though isolation of pure cultures was not successful. One nitrate-reducing cysteate-oxidizing bacterium, strain NKNCYSA, was studied in detail. It was identified as Paracoccus pantotrophus. Eighteen sulfonates were tested, and the organism degraded cysteate, taurine, isethionate (2-hydroxyethanesulfonate), sulfoacetate or 3-amino-propanesulfonate with concomitant reduction of nitrate, presumably to molecular nitrogen. The carbon skeleton of these substrates was converted to cell material and, presumably, CO2. The amino group was released as ammonia and the sulfono moiety was recovered as sulfate. Cell-free extracts of P. pantotrophus NKNCYSA contained constitutive L-cysteate:2-oxoglutarate aminotransferase (EC 2.6.1.-) and glutamate dehydrogenase (EC 1.4.1.4). Taurine:pyruvate aminotransferase, in contrast, was inducible.
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Possible involvement of cAMP in aerial mycelium formation and secondary metabolism in Streptomyces griseus
More LessIn Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) triggers secondary metabolism and morphogenesis by binding a repressor protein (ArpA) and dissociating it from DNA. UV-mutagenesis of the A-factor-deficient mutant HH1 generated strain HO2, defective in the synthesis of ArpA and therefore able to form aerial mycelium, spores and streptomycin. Shotgun cloning of chromosomal DNA from wild-type S. griseus in strain HO2 yielded a gene that suppressed aerial mycelium formation and streptomycin production. Nucleotide sequencing and subcloning revealed that the gene encoded a eukaryotic-type adenylate cyclase (CyaA). In mutant HO2 production of cAMP was growth-dependent until the middle of the exponential growth stage; the production profile was the same as in the wild-type strain. However, the amount of cAMP produced was five times larger when mutant HO2 harboured cyaA on the high-copy-number plasmid plJ486. Consistent with this, supplying cAMP exogenously at a high concentration to mutant HO2 suppressed formation of both aerial mycelium and streptomycin. On the other hand, some lower concentrations of cAMP stimulated or accelerated aerial mycelium formation. No effects of exogenous cAMP on morphogenesis and secondary metabolism were apparent in the wild-type strain. In addition, disruption of the chromosomal cyaA gene in the wild-type strain had almost no effect. Introducing cyaA cloned in either a low- or a high-copy-number plasmid suppressed morphogenesis and secondary metabolism not only in mutant HO2 but also in other arpA mutants, implying that the effects of cAMP became apparent in the arpA-defective background. When mutant HO2 carried cyaA on a plasmid, synthesis of the stringent response factor ppGpp was greatly reduced; this may account for the observed suppression by cAMP of morphogenesis and secondary metabolism. cAMP also affected protein tyrosine phosphorylation, as determined with anti-phosphotyrosine antibody.
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Butane metabolism by butane-grown ‘Pseudomonas butanovora’
More LessThe pathway of butane metabolism by butane-grown ‘Pseudomonas butanovora' was determined to be butane → 1-butanol → butyraldehyde → butyrate. Butane was initially oxidized at the terminal carbon to produce 1-butanol. Up to 90% of the butane consumed was accounted for as 1-butanol when cells were incubated in the presence of 5 mM 1-propanol (to block subsequent metabolism of 1-butanol). No production of the subterminal oxidation product, 2-butanol, was detected, even in the presence of 5 mM 2-pentanol (an effective inhibitor of 2-butanol consumption). Ethane, propane and pentane, but not methane, were also oxidized. Butane-grown cells consumed 1-butanol and other terminal alcohols. Secondary alcohols, including 2-butanol, were oxidized to the corresponding ketones. Butyraldehyde was further oxidized to butyrate as demonstrated by blocking butyrate metabolism with 1 mM sodium valerate. Butyrate also accumulated from butane when cells were incubated with 1 mM sodium valerate. The pathway intermediates (butane, 1-butanol, butyraldehyde and butyrate) and 2-butanol stimulated O2 consumption by butane-grown cells. 1-Butanol, butyraldehyde and butyrate supported growth of ‘P. butanovora’, as did 2-butanol and lactate.
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- Plant-Microbe Interactions
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A unique eukaryotic β-xylosidase gene from the phytopathogenic fungus Cochliobolus carbonum
More LessThe plant-pathogenic fungus Cochliobolus carbonum secretes one major β-xylosidase (Xyp1) when grown on xylan or maize cell walls. cDNA and genomic DNA encoding Xyp1 were isolated using PCR primers based on peptide sequences from the purified protein. XYP1 contains three introns, has 5' and 3' untranslated regions of 74 and 145 bp, respectively, and is predicted to encode a protein of 328 amino acids (M r 36 700) with four N-glycosylation sites. Although it is secreted, Xyp1 has no predicted signal peptide. Furthermore, Xyp1 appears not to be processed at the N-terminus because one of the peptides isolated from the mature protein is located only six amino acids downstream of the translational start methionine. The primary sequence of Xyp1 is unrelated to any known eukaryotic β-xylosidase but has 35% overall identity to two bacterial bifunctional β-xylosidase/α-arabinosidases. Mutation of XYP1 by targeted gene replacement resulted in the loss of the major β-xylosidase activity corresponding to the product of XYP1, but a significant amount of secreted β-xylosidase activity (25% of wild-type) remained in the culture filtrates. The xyp1 mutant was still fully pathogenic on maize.
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- Systematics And Evolution
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Towards understanding the evolution of the human commensal yeast Candida albicans
More LessAllelic frequencies and relationships for one dimorphic locus and three unlinked polymorphic loci have been determined for 114 unrelated isolates of Candida albicans, including 14 laboratory reference strains and 50 strains from each of two geographic regions. Although there was no indication of geographical partitioning, there were significant correlations for specific allelic pairs among loci and little evidence that any alleles were in Hardy-Weinberg equilibrium. This gives additional support for the concept that the primary mode of genetic inheritance in this species is clonal, with other intracellular genetic events playing a lesser role in the creation of genomic diversity. Through inference of this and other known attributes of closely related Candida species, such as sequence analysis of IS1 and the ITS2 (internal transcribed spacer 2) region of the rDNA cistron, the deduced phylogeny suggests an evolutionarily recent origin for many frequently isolated strains. This finding will be of interest in the context of understanding pathogenicity and drug resistance in this human commensal yeast.
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