- Volume 140, Issue 12, 1994
Volume 140, Issue 12, 1994
- Review Article
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- Sgm Special Lecture
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- Microbiology Comment
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- Antigens And Immunity
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Detection of Penicillium aurantiogriseumby ELISA utilizing antibodies produced against its exoantigens
More LessAn indirect competitive ELISA was developed using rabbit antiserum against the exoantigens (ExAgs) of Penicillium aurantiogriseum, and cross-reactivities were determined with 15 ExAgs from other species and genera of fungi and with the water-soluble extracts from four grains. The assay had good sensitivity to P. aurantiogriseumExAgs (1 μg per ml serum), with generally low or no cross-reactivity with grain extracts and the other fungi, including five species of Penicillium, four species of Aspergillus, three species of Fusarium, two species of Mucorand one species of Alternaria.Immunoblotting analysis of the ExAgs from P. aurantiogriseumyielded at least 20 distinct antigenic bands, with about 3 or 4 being dark, 10-12 light and 5-8 faint in colour. Most of the bands had molecular masses of about 70-90 kDa. In contrast to P. aurantiogriseum, the ExAgs from the other fungi either did not react in the immunoblotting assay with the antiserum, or reacted only weakly with it. One antigenic band from P. roqueforti, however, reacted moderately strongly with the antiserum. The ELISA and immunoblotting analysis were used to detect P. aurantiogriseumin naturally contaminated wheat samples, and in wheat samples spiked with a second common storage fungus, Aspergillus ochraceus.The results demonstrated that the antiserum was able to detect P. aurantiogriseumin a background of other fungal species, and that these did not produce false positives. There was also a positive association between the concentration of P. aurantiogriseumExAgs, as measured by ELISA, and the amount of mould as determined by the number of colony-forming units. Immunoblotting qualitatively confirmed the ELISA results obtained with fungi when cultured on liquid or on solid (wheat) media. The data suggest that the immunoassays developed for P. aurantiogriseumare useful for the detection and identification of P. aurantiogriseumon the basis of its ExAgs, with the advantages of being more efficient, simple and reliable than conventional techniques.
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Comparison of outer-membrane proteins of Pasteurella haemolyticaexpressed in vitroand in vivoin cattle
More LessOuter-membrane protein (OMP) profiles of two serotype A1 isolates of Pasteurella haemolyticawere compared by SDS-PAGE and Western blotting with bovine convalescent serum after growth (a) in vitrounder iron-sufficient and -deficient conditions, (b) in vivoin the lungs of experimentally infected calves and (c) in vivoin diffusion chambers implanted into the peritoneal cavities of calves. Lung-grown bacteria differed from iron-sufficient in vitro-grown bacteria in having enhanced expression of the previously recognized 71, 77 and 100 kDa iron-regulated proteins, reduced expression of 18, 31, 39.5 and 50 kDa proteins, and expression of a 19 kDa protein. Differences were also apparent in the Western blot profiles of OMPs of in vitro- and lung-grown bacteria. These included the apparent lack of recognition of the 100 kDa protein in the lung-grown bacteria, but not in the in vitro-grown bacteria, and more intense staining of a 47 kDa protein in in vitro-grown bacteria, but not in lung-grown bacteria. The OMP profiles of the chamber-grown bacteria resembled those of the lung-grown bacteria in that expression of the 18, 19, 31 and 39.5 kDa proteins was similar. These similarities demonstrated that the chamber-grown bacteria had adapted to the in vivoenvironment, and that growth conditions within the chambers resembled, but not perfectly, those within the lungs. For example, expression of the three iron-regulated OMPs was very low in the chamber-grown bacteria compared to the lung-grown bacteria. The OMP profiles of bacteria grown in vitroin newborn calf serum closely resembled those of lung-grown bacteria, suggesting that in vivogrowth may be partly reproduced in vitroby growing the bacteria in newborn calf serum.
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- Biochemistry
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Pectin methylesterase from Botrytis cinerea: physiological, biochemical and immunochemical studies
More LessPectin methylesterase (PME) was purified from the supernatant of Botrytis cinereastrain Bd90. SDS-PAGE showed a single band at 42 kDa, but this band corresponded to two distinct isoforms observed by IEF-PAGE at pl values of 7.0 and 7.4. PME was produced during the exponential phase of fungal growth and independently of the carbon source. Unlike other pectinases of B. cinerea, which are polymorphic, no differences were observed between the PME profiles of 25 strains of different origins. Polyclonal antibodies were raised against purified PME from B. cinerea, and immunochemical comparisons with PMEs from Erwinia chrysanthemi, Vigna radiataand Glycine maxshowed the presence of common epitopes between these different enzymes.
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Analysis of different DNA fragments of Corynebacterium glutamicumcomplementing dapEof Escherichia coli
More LessIn Corynebacterium glutamicum L-lysine is synthesized simultaneously via the succinylase and dehydrogenase variant of the diaminopimelate pathway. Starting from a strain with a disrupted dehydrogenase gene, three different-sized DNA fragments were isolated which complemented defective Escherichia colimutants in the succinylase pathway. Enzyme studies revealed that in one case the dehydrogenase gene had apparently been reconstituted in the heterologous host. The two other fragments resulted in desuccinylase activity; one of them additionally in succinylase activity. However, the physical analysis showed that structural changes had taken place in all fragments. Using a probe derived from one of the fragments we isolated a 3.4 kb BamHl DNA fragment without selective pressure (by colony hybridization). This was structurally intact and proved functionally to result in tenfold desuccinylase overexpression. The nucleotide sequence of a 1966 bp fragment revealed the presence of one truncated open reading frame of unknown function and that of dapEencoding N-succinyl diaminopimelate desuccinylase (EC 3.5.1.18). The deduced amino acid sequence of the dapEgene product shares 23% identical residues with that from E. coli.The C. glutamicumgene now available is the first gene from the succinylase branch of lysine synthesis of this biotechnologically important organism.
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Characterization of the major catalase from Streptomyces coelicolorATCC 10147
More LessStreptomyces coelicolorATCC 10147 produced catalases whose electrophoretic mobility varied depending on the growth phase in liquid culture. Polyacrylamide gel electrophoresis of cell extracts resulted in six catalase activity bands, which were designated Cat1 to Cat6. Of these. Cat4 appeared during all growth phases, whereas Cat1 appeared only during the stationary phase. Catalase-deficient mutants were screened by the H2O2bubbling test following NTG mutagenesis. In all the non-bubbling mutants tested, the Cat4 activity band significantly decreased or disappeared, suggesting that Cat4 is the major catalase. Cat4 was purified to electrophoretic homogeneity and some of its properties analysed. The enzyme has a native molecular mass of 225 kDa, as determined by gel permeation column chromatography, and consists of four identical subunits of 57 kDa, as determined by SDS-PAGE. The enzyme contains 2.6 molecules of protohaem IX per tetramer, as indicated by the absorption spectrum. It was not reducible by sodium dithionite and exhibited no peroxidase activity with o-dianisidine as the substrate. All these characteristics, as well as inhibitor studies, indicate that the major vegetative catalase in S. coelicolor, unlike E. colivegetative catalase, is a member of the typical monofunctional catalases found in eukaryotes and some bacteria.
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- Development And Structure
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Induction of myxospores in Stigmatella aurantiaca(myxobacteria): analysis of inducer-inducer and inducer-inhibitor interactions by dose-response curves
More LessTheories and methods developed in molecular pharmacology for drug receptor interactions were used to explain artificially induced myxospore formation. The correlation between inducer concentrations and the yield of myxospores, i.e. the experimentally obtained dose-response curves, were much steeper than expected for an interaction based on mass action law. We postulate that the interaction of an inducer with one of the different receptors of the bacterial cell causes the production of a common stimulus which programmes the cells to turn into myxospores, if a threshold value is reached. Simultaneous addition of inducers of the same or of different inducer groups showed synergistic interactions. Inducer concentrations that by themselves were not inducing gave maximum myxospore formation when combined. Addition of pyrrole, a specific inhibitor, caused a concentration-dependent parallel shift of the glycerol dose-response curve. Compounds, inducers or inhibitors, with identical receptor specificities competed for the common receptor according to their affinity. Interactions of inducers with different receptor specificities could be predicted from calculations with a mathematical model of functional synergism.
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Burkholderia(formerly Pseudomonas) cepaciaporin is an oligomer composed of two component proteins
More LessThe 81 kDa protein (designated OpcPO) which forms a diffusion pore in the outer membrane of Burkholderia(formerly Pseudomonas) cepaciahas a unique characteristic in that when the purified protein is heated it yields a major 36 kDa protein (designated OpcP1) and a minor 27 kDa protein (designated OpcP2). Moreover, incubation of OpcPO in citrate buffer at pH 3.0 produced an unusual dissociation into 72 kDa and 27 kDa proteins. For the characterization of OpcPO and its derivatives, OpcP1 and OpcP2 from purified OpcPO were isolated by preparative SDS-PAGE. Reconstitution of OpcPO using purified preparations of OpcP1 and OpcP2 indicated that these derivatives were not proteolytic fragments of OpcPO. Moreover, immunoblot assays with murine polyclonal antisera specific for OpcP1 and OpcP2 yielded the following results: (i) OpcP1 and OpcP2 are immunologically distinguishable proteins; (ii) the unusual dissociation of OpcPO in citrate buffer at pH 3.0 resulted in the release of OpcP2 from OpcPO, and the resulting 72 kDa protein was probably an oligomer of OpcP1; (iii) purified OpcP1 itself produced two additional 53 kDa and 72 kDa proteins spontaneously following elution from the bottom of the SDS-PAGE gel. From these findings, it was concluded that OpcPO is formed by the non-covalent association of OpcP2 with an oligomer of OpcP1 that has the ability to self-assemble.
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- Environmental Microbiology
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Phosphorylation of membrane proteins in response to temperature in an Antarctic Pseudomonas syringae
More LessTemperature-dependent phosphorylation and dephosphorylation of membrane proteins was studied in vitroin a number of psychrotrophic Antarctic bacteria which grow between 0 and 30°C. One of them, a Pseudomonas syringaeisolate, was studied in detail and was found to have three membrane proteins of molecular mass 30, 65 and 85 kDa which were phosphorylated differently in response to low and high temperatures. The 65 kDa protein was phosphorylated only at lower temperatures (between 0 and 15°C). The 30 kDa protein was phosphorylated more at higher temperatures and was possibly a histidine kinase. This protein was present in all the psychrotrophic Pseudomonasspecies studied and in Sphingobacterium antarcticus.A possible role for these proteins in sensing environmental temperature is proposed.
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- Genetics And Molecular Biology
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Elements interrupting nitrogen fixation genes in cyanobacteria: presence and absence of a nifDelement in clones of Nostocsp. strain Mac
More LessNostocsp. strain Mac is capable of microaerobic, but not aerobic, nitrogen fixation (Fox). NostocMac grows as long, relatively straight, filaments that are well dispersed in the culture medium. However, spontaneouslγ-arising revertant strains selected for aerobic nitrogen fixation (Fox+) all grow as coiled filaments that associate in macroscopic clumps or balls of varying dimensions. DNA restriction fragment length polymorphism, using nitrogenase (nif) structural genes as probes, established identity between revertants and the parental culture. Mapping of the fragments and lack of hybridization to specific probes indicated the absence of a DNA sequence interrupting the nifDgene in one Fox+revertant. Such a nifDelement is assumed to be present in essentially all heterocyst-forming cyanobacteria. Only one clone out of 223 Fox-and Fox+ NostocMac clones surveyed lacked the nifDelement, indicating that loss of the element is a rare event. The nifDelement is present in the same location in the genome of NostocMac as it is in all other heterocyst-forming cyanobacteria analysed. No phenotypic differences could be detected between two Fox+clones containing or lacking the nifDelement, including repression and derepression of nitrogen fixation in response to the presence or absence of combined nitrogen. We suspect that retention of the nifDelement in vegetative cells of heterocyst-forming cyanobacteria is a consequence of selective pressure, although such selective conditions in laboratory cultures have not been identified.
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Transposon mutagenesis of Nostocsp. strain ATCC 29133, a filamentous cyanobacterium with multiple cellular differentiation alternatives
More LessNostocsp. strain ATCC 29133 (PCC 73102; Nostoc29133) is a symbiotically-competent, facultatively heterotrophic, diazotrophic cyanobacterium with the capacity to differentiate specialized cells such as heterocysts, akinetes and hormogonial filaments. We have optimized several methods for physiological and molecular genetic analysis of Nostoc29133. By use of a Tn5derivative, Tn5-1063 (KmrBmrSmr), delivered by conjugation from Escherichia coli, antibiotic-resistant mutants of Nostoc29133 were generated at a frequency of approximately 1 × 10−6, 0.4% of which expressed a nitrogen fixation (heterocyst) defective phenotype. Mutant strain UCD 328 was isolated after co-culture of 86 Nostoc29133::Tn5-1063 clones with the symbiotic plant partner, Anthoceros punctatus; strain UCD 328 expressed a symbiotic phenotype of increased frequency of hormogonia-dependent infection. The transposon and flanking genomic DNA was recovered from strain UCD 328, the mutation and phenotype reconstructed by homologous recombination in Nostoc29133, and the transposition site identified from a Nostoc29133 genomic library. Transposon mutagenesis has thus provided the means for isolation and identification of developmental and symbiotic-specific genes of Nostoc29133.
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Analysis of Saccharomyces cerevisiaeproteins induced by peroxide and superoxide stress
More LessExponentially growing Saccharomyces cerevisiaecells are more sensitive to oxidants such as hydrogen peroxide and superoxides than stationary phase cells. Using disruption mutations in the genes encoding the two S. cerevisiaesuperoxide dismutases, we show that the principal mechanism of toxicity of redox-cycling compounds, such as menadione and plumbagin, is via the production of superoxide anions. Using two-dimensional polyacrylamide gel electrophoresis we have compared the pattern of protein expression in cells labelled with L-[35S]methionine and stressed with either H2O2or menadione. Three groups of proteins were evident: those whose levels are elevated by both H2O2and menadione, and those specifically induced by either H2O2or menadione. Experiments with promoter fusions demonstrated that one of the heat inducible forms of HSP70 (SSA1) was inducible with H2O2. Furthermore, induction of the yeast H2O2-responsive TRX2promoter by menadione required the metabolism of menadione.
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Occurrence of chromosome rearrangements during the fusion process in the imperfect yeast Candida albicans
More LessAuxotrophic derivatives of three strains of the pathogenic yeast Candida albicansof different origins, including 1006 derived from CBS5736, A5153 derived from FC18 and NARA2 derived from NUM961, were used in spheroplast fusion experiments. The DNA content of the prototrophic fusion product obtained following fusion between strains 1006 and A5153 approximated to the sum of those of the parents, but was variable when NARA2 was used as the parent for fusion. Chromosome-sized DNA molecules of the fusion derivatives were separated by pulsed-field gel electrophoresis to examine whether either or both of the chromosome-sized DNA molecules of each parent were transferred into the fusion derivatives. In the fusion derivatives obtained following fusion between strains 1006 and A5153, nearly the full complement of chromosomes was shown to be transferred, but partial transfer of chromosomes occurred in the fusion derivatives that were obtained following fusion between strains NARA2 and A5153. Results indicated that chromosome loss also occurred when these two strains were fused. Variations in the size of R chromosomes, the rDNA-containing chromosomes, were observed in all fusion derivatives tested, indicating high-frequency recombination between R chromosomes during the fusion process.
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Cloning and expression in Escherichia coliof DNA encoding a 60 kDa stress protein of Mycobacterium paratuberculosis, the causative agent of Johne's disease
More LessPolymerase chain reaction (PCR) was used to generate DNA encoding a 60 kDa stress protein of Mycobacterium paratuberculosisusing primers complementary to sequences at the 5′ and 3′ ends of 60 kDa stress protein genes (encoding the ‘65 kDa antigens’) of M. lepraeand M. tuberculosis.The predicted PCR product of 1.8kb contained the entire coding sequence of an M. paratuberculosis60kDa stress protein, with non-coding regions of 124bp and 1bp at the 5′ and 3′ ends, respectively. DNA encoding the entire ORF for the 60kDa stress protein, as well as thrombin and Factor Xa proteolytic cleavage sites, was ligated into the bacterial expression vector pGEX-2T and used to transform Escherichia colistrain JM83. Transformed bacteria, induced by IPTG, expressed an 85 kDa fusion protein comprising glutathione S-transferase (GST) and M. paratuberculosis60 kDa stress protein. This fusion protein was purified by adsorption to glutathione-agarose beads and shown to cross-react in Western blot analysis with an anti-mycobacterial 60 kDa stress protein monoclonal antibody. Recombinant M. paratuberculosis60 kDa stress protein was liberated from GST by proteolytic cleavage with either thrombin or Factor Xa enzyme. Authenticity of liberated recombinant stress protein was confirmed by N-terminal amino acid sequencing.
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The Escherichia coli dsbAgene is partly transcribed from the promoter of a weakly expressed upstream gene
More LessThe dsbAgene of Escherichia coliencodes a periplasmic enzyme which catalyses disulfide bond formation. Analysis of its surrounding DNA region showed that it is preceded by an open reading frame, orfA, of 984 nucleotides. The intergenic region (19 nucleotides) carries no typical transcription termination signals. dsbAis transcribed from two promoters, the first (P1) lies in the distal part of orfA, and the second (P2) just upstream from orfA. Using a plasmid-borne dsbA:: TnphoAfusion and an orfA:: ω insertion, each promoter was shown to contribute equally to dsbAtranscription. The disruption of the single chromosomal copy of orfAby ω more drastically reduced the amount of DsbA in the periplasmic space. Such a reduction of the DsbA pool, however, did not change the activities of the AppA, Agp and PhoA periplasmic phosphatases, which all require disulfide bond formation, even when the enzymes were produced from multicopy recombinant plasmids. Thus, in a wild-type strain, DsbA is far from being in limiting amounts for physiological requirements. The orfAgene product was identified as a weakly expressed 39 kDa cytoplasmic protein, but it is not involved in the overall mechanism of disulfide bond formation.
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Transcription analysis of the Streptomyces coelicolorA3(2) rrnAoperon
Transcription start sites and processing sites of the Streptomyces coelicolorA3(2) rrnAoperon have been investigated by a combination of in vivoand in vitrotranscription analyses. The data from these approaches are consistent with the existence of four in vivotranscription sites, corresponding to the promoters P1-P4. The transcription start sites are located at −597, −416, −334 and −254 relative to the start of the 16S rRNA gene. Two putative processing sites were identified, one of which is similar to a sequence reported earlier in S. coelicolorand other eubacteria. The P1 promoter is likely to be recognized by the RNA polymerase holoenzyme containing σhrdB, the principal sigma factor in S. coelicolor.P2 also shares homology with the consensus for vegetative promoters, but has a sequence overlapping the consensus −35 region that is also present in the −35 regions of P3 and P4. The −35 sequence common to P2, P3 and P4 is not similar to any other known consensus promoter sequence. In fast-growing mycelium, P2 appears to be the most frequently used promoter. Transcription from all of the rrnApromoters decreased during the transition from exponential to stationary phase, although transcription from P1 and P2 ceased several hours before that from P3 and P4.
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Possible involvement of the lysine ε-aminotransferase gene (lat) in the expression of the genes encoding ACV synthetase (pcbAB) and isopenicillin N synthase (pcbC) in Streptomyces clavuligerus
Streptomyces clavuligerusproduces the β-lactam antibiotics penicillin N, O-carbamoyldeacetycelcephalosporin C and cephamycin C. We characterized a wild-type DNA region which restores antibiotic formation to a mutant strain named NP1, previously shown to exhibit depressed activities for two early enzymes of cephalosporin synthesis, δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (IPNS). L-Lysine ε-aminotransferase (LAT) assays and α-AAA feeding experiments suggested that strain NP1 is a latmutant. NP1 recovered LAT, ACVS and IPNS activities when transformed with the cloned region. DNA sequencing showed that this region encodes the entire LAT gene (lat), required for the conversion of L-lysine to the β-lactam precursor L-α-aminoadipic acid (α-AAA), as well as the upstream half of the ACVS gene (pcbAB). The activities of ACVS and IPNS appear to depend upon LAT expression. Gene fusions constructed to investigate promoter activities in the cloned region support a model of interdependence in the expression of the genes for LAT, ACVS and IPNS (pcbC).
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Cloning and characterization of polyketide synthase genes for jadomycin B biosynthesis in Streptomyces venezuelaeISP5230
More LessHybridizing fragments in the genomic DNA of Streptomyces venezuelaeISP5230, which produces the jadomycin group of angucycline antibiotics, were detected by probing with actlDNA from Streptomyces coelicolorA3(2). The hybridizing regions were isolated from a 16.5 kb insert of S. venezuelae DNA recovered from a genomic library cloned in aλ replacement vector. Subcloning and sequencing of a 4.8 kb segment of the insert, containing regions hybridizing to actIIIas well as actl, identified five open reading frames (ORFs). The deduced polypeptide products of the ORFs closely resemble in sequence the components of streptomycete type-II polyketide synthases (PKSs): the ORF1 product corresponds to the ketoacyl synthase, and the ORF2 product to a polypeptide closely related to the ketoacyl synthase and involved in determining chain length; the ORF3 product matches the acyl carrier protein; ORF4 encodes a bifunctional cyclase/dehydrase; and ORF5 encodes a ketoreductase. Integration into the chromosomal DNA of a plasmid containing a segment of the ORF2-ORF4 region severely depressed jadomycin B biosynthesis; since the integrant showed no change in growth or spore pigmentation, the cloned PKS genes are presumed to encode enzymes in the pathway for jadomycin biosynthesis.
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- Pathogenicity And Medical Microbiology
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Expression of the Bordetella pertussisP.69 pertactin adhesin in Escherichia coli: fate of the carboxy-terminal domain
The mature pertactin protein (P.69) of Bordetella pertussiscan be isolated from the bacterial cell surface as a polypeptide with an apparent molecular mass of 69000 Da as determined by sodium dodecyl sulphate gel electrophoresis. However the open reading frame of prn, the pertactin gene, encodes a polypeptide with a predicted molecular mass of 93478 Da, referred to as P.93. Expression of the prngene in Escherichia colileads to the synthesis of the full-length P.93 polypeptide, which is rapidly processed to the mature P.69 protein located at the cell surface. The P.93 precursor polypeptide is processed at both termini. A 34 amino acid long signal sequence is removed from the amino-terminus and a polypeptide sequence of about 30000 Da (P.30) is cleaved from the carboxy-terminus. Deletion of the 3′ region of prn, encoding P.30, results in the expression of an intracellular form of P.69. Antiserum which recognizes P.30 was raised using synthetic peptides based on the primary amino acid sequence of the region. This anti-P.30 serum was used in a Western blot analysis of fractionated cells of B. pertussisand E. coliharbouring the intact prngene. The P.30 polypeptide was readily detected in outer membrane fractions prepared from both of these bacterial species, although it could not be shown to be exposed at the cell surface.
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Serum-sensitive mutation of Francisella novicida: association with an ABC transporter gene
Francisella novicidais a facultative intracellular pathogen that can survive and grow in macrophages by preventing phagolysosomal fusion. In this study in vitrocassette mutagenesis was used to generate a library of insertion mutants of F. novicida.Two related mutants, KM14 and KM14S, initially identified as defective for growth in macrophages, were found to be sensitive to serum. These mutants were also found to grow approximately 1000-fold less well in the livers and spleens of infected mice. We cloned a genetic locus that was presumably mutagenized in these mutants and found that it included genes that had high similarity in their deduced amino acid sequence to those of msbAand orfEof Escherichia coli.The former is a member of the superfamily of ABC transporter proteins. We named the corresponding genes in F. novicida, valAB.Integration of a cloned valABlocus into the chromosome of KM14S partially restored the serum resistance phenotype found in wild-type F. novicida.
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Cloning and expression in Escherichia coliof the nahAgene from Porphyromonas gingivalisindicates that β-N-acetylhexosaminidase is an outer-membrane-associated lipoprotein
More LessPorphyromonas gingivalishas been implicated in human periodontal diseases. It expresses a number of exoglycosidase enzymes capable of hydrolysing host proteoglycan residues. As a first stage to explore the role of these enzymes in periodontal tissue damage, the nahAgene of P. gingivalisW83, which encodes β-N-acetylhexosaminidase (β-Nahase), was cloned. The gene was expressed poorly in Escherichia coli, but increased expression was achieved by cloning the nahAgene downstream of the tac promoter. Southern blot analysis revealed that nahAwas present as a single copy, and it was found in all the other P. gingivalisstrains tested. In contrast, sequences homologous to nahAwere not detected in either P. endodontalisor P. asaccharolytica.The nahAgene was 2331 bp long and encoded a β-Nahase enzyme of 777 amino acids with a predicted molecular mass of 87 kDa. A characteristic signal peptide for an acylated lipoprotein was present at the amino-terminus, suggesting that the mature β-Nahase is a lipoprotein. The predicted amino acid sequence of the P. gingivalisβ-Nahase shared homology with the catalytic domains of the human β-Nahase enzyme and the chitinase of Vibrio harveyi, suggesting a common catalytic mechanism.
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- Physiology And Growth
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Studies of the biosynthesis of tentoxin by Alternaria alternata
More LessBiosynthesis of the phytotoxin, tentoxin, its regulation and the enzymic synthesis steps were studied in vivoand in vitro.The physiology of biosynthesis of tentoxin in vivowas investigated by using sections of mycelial mats incubated in buffer. Differentiated mycelia could be studied under defined conditions. The de novosynthesis of tentoxin was measured by incorporation of [U-14C]leucine into tentoxin. The investigation system was stable for 10 h. Biosynthesis and the growth of biomass started before day 5 of culture, with the maximum between days 9 and 12. After this, biosynthesis quickly declined. pH values about 7 were optimal, and pH values above and below this led to an increased release of tentoxin stored in the cells. The formation of tentoxin by older mycelia was not regulated by acetate, phosphate or glucose, which was not utilized. Precursor amino acids, applied at the start of the culture, slightly activated the synthesis of tentoxin. Older mycelia were inhibited. Substances from the host plant (Brassica chinensis) reduced the de novosynthesis of tentoxin. Enzyme separation studies suggested that biosynthesis of tentoxin involves a multienzyme (≥ 400 kDa), which is a polyfunctional protein without subunits. Experiments suggested that the synthetase contains active SH-groups and an integrated activity of methyltransferase. The precursor amino acids are activated by ATP and bound at the enzyme. N-Methylation occurs with the enzyme-bound amino acids or during the elongation of the growing peptide chain. Methionine is the primary donor of the methyl groups, but the immediate methylation reaction needs S-adenosyl methionine (SAM). The methylation is essential for the continuation of biosynthesis. The elongation proceeds either stepwise from glycine by binding alanine/methylalanine, phenylalanine/methylphenylalanine and leucine or by formation and linkage of two dipeptides glycine-alanine/methylalanine and phenylalanine/methylphenylalanine-leucine. At the end of this process dihydrotentoxin, the direct precursor of tentoxin, is released from the synthetase probably by cyclization. Independent of this first enzyme, dihydrotentoxin is transformed into tentoxin. This last reaction step is reversible. The rate of transformation of dihydrotentoxin to tentoxin is higher, but in this direction the native turnover is relatively low. The synthesis of tentoxin probably occurs in a manner similar to other well-known cyclic peptides via a ‘thiotemplate mechanism’; the highest enzyme activity in vitrooccurs between days 9 and 11 of culture at a pH value of 7.
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Metabolic cooperation in oral microbial communities during growth on mucin
More LessHog gastric mucin has been used as a model glycoprotein to determine the role of particular glycosidases produced by different oral bacteria in the development of stable, diverse microbial communities. The patterns of glycosidase and protease activity were determined in pure cultures of ten representative species of oral bacteria using synthetic substrates. A five member mixed culture was established in a chemostat, comprising species with minimal glycosidase and protease activity, in which hog gastric mucin was the major carbon and energy source. Introduction of additional species with novel enzyme activities (e.g. sialidase, α-fucosidase and endopeptidase) led to their establishment within the community to make communities with seven, eight, nine and ten members and resulted in an increase in the total viable counts of the microbial consortium. This increase in viable count was made up of the numbers of the newly added species as well as from a rise in the numbers of the existing community members. This result suggested that glycoprotein catabolism involved the synergistic and concerted action of several species with overlapping patterns of enzyme activity. Such metabolic cooperation results in the liberation of additional nutrients, and this may help to maintain the characteristic diversity of resident microbial communities found in many natural habitats.
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The influence of ionic strength and pH on diffusion of micro-organisms with different structural surface features
More LessExact knowledge of microbial diffusion coefficients is a prerequisite for the application of mass transport theories to microbial deposition data. Microbial diffusion coefficients can be calculated on the basis of cell radii using the Einstein equation. This approach, however, does not take into account the additional effects of structural cell surface features such as fibrils or a fuzzy coat. Microbial diffusion coefficients of micro-organisms in suspension can be experimentally measured using dynamic light scattering. In this paper we compare experimental microbial diffusion coefficients with those calculated from radii for a variety of microbial strains suspended in 10 mM or 40 mM potassium phosphate solutions of different pH. Furthermore, the pH dependence of the microbial diffusion coefficients is related to the pH dependence of the microbial zeta potentials in similar ionic strength solutions. Microbial diffusion coefficients ranged from 2 × 1013to 5 × 1013 m2s−1and were generally higher at low pH (pH 2) than at high pH (pH 7) for strains with structural surface features. Experimental diffusion coefficients at pH 2 were approximately similar to those based on microscopic radii. This indicates that at pH 2 structural surface features are in a collapsed state, presumably due to the lack of stabilizing electrostatic repulsion between the cell surface structures as shown by the zeta potentials measured. Alternatively, a hydrodynamic radius of a micro-organism can be calculated from the experimental diffusion coefficients and the Einstein equation. Assuming that the maximal difference in hydrodynamic radius, observed over the pH range employed, represents the maximal length of structural cell surface features present, then the calculated fibrillar lengths for Streptococcus mitisBA and S. salivariusHB are in reasonable agreement with electron microscopical estimates.
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Regulation of 2-deoxyglucose phosphate accumulation in Lactococcus lactisvesicles by metabolite-activated, ATP-dependent phosphorylation of serine-46 in HPr of the phosphotransferase system
More LessLactococcus lactistakes up glucose and the nonmetabolizable glucose analogue 2-deoxyglucose (2DG) via the phosphotransferase system and extrudes the accumulated sugar phosphates in a process apparently dependent on a cytoplasmic sugar-phosphate phosphatase. Uptake of 2DG into L. lactisvesicles was shown to be dependent on an energy source, effectively provided by intravesicular phosphoenolpyruvate (PEP). 2DG phosphate (2DG-P) accumulation in these vesicles was not inhibited, and preaccumulated 2DG-P was not released from them, upon electroporation of fructose 1,6-diphosphate (FDP), gluconate 6-phosphate or 2-phosphoglycerate into the vesicles. Intravesicular but not extravesicular wild-type HPr of Bacillus subtilisalone stimulated uptake, but in the presence of any one of these metabolites, it prevented accumulation of 2DG-P. Intravesicular H15A mutant HPr inhibited uptake and allowed further inhibition of 2DG-P accumulation in the presence of the intravesicular metabolites. Intravesicular S46A mutant HPr stimulated uptake but could not promote inhibition in the presence of the phosphorylated metabolites. The S46D mutant HPr protein promoted regulation, even in the absence of a metabolite. The V maxbut not the K mvalue for 2DG uptake was affected. Accumulation of the natural, metabolizable substrates of the lactose, glucose, mannose and ribose permeases was inhibited by wild-type HPr in the presence of FDP or by S46D mutant HPr. The results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase selectively determines the levels of sugar accumulation via the glucose and lactose permeases in L. lactis.They suggest that two primary functions of HPr(Ser) phosphorylation are: (1) to feedback-inhibit the activities of carbohydrate permeases and not merely to create a hierarchy of preferred carbon sources, and (2) to regulate the cytoplasmic concentrations of carbohydrate inducers by exclusion and expulsion mechanisms.
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- Systematics
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The dynamic progression of evolved character states for aromatic amino acid biosynthesis in Gram-negative bacteria
More LessA systematic analysis of the evolution of aromatic amino acid biosynthesis in the Proteobacteria, previously focussed mainly upon the γ subdivision, has now been extended to the β subdivision. Five lineages were studied, represented by Neisseria gonorrhoeae, Nitrosomonas europaea, Alcaligenes faecalis, rRNA Group-III pseudomonads/Rubrivivax gelatinosus, and rRNA Group-II pseudomonads/Rhodocyclus tenuis.Within the phenylalanine pathway, the bifunctional P-protein (chorismate mutase/prephenate dehydratase) was present in each lineage and must have evolved in a common ancestor of the β and γ subdivisions. Each P-protein was found to be subject to activation by L-tyrosine, and to feedback inhibition by L-phenylalanine. Phenylalanine-inhibited (DS-phe) and tyrosine-inhibited (DS-tyr) isoenzymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase probably existed in the common β-subdivision ancestor, with DS-tyr being lost in N. gonorrhoeaeand A. faecalis.The participation of DS-phe in a dissociable multienzyme complex with one or more other common-pathway enzymes is known to exist in N. gonorrhoeae.The same complex is indicated by two peaks of DS-phe seen in chromatographic profiles of Group-III pseudomonads and A. faecalis.It is concluded that the contemporary DS-phe species present in subdivisions γ and β must have had independent origins. Tyrosine biosynthesis was found to be quite diverse within the β subdivision. Nit. europaeapossessed an arogenate dehydrogenase which was specific for NADP+. In all other lineages, a broad-specificity cyclohexadienyl dehydrogenase (CDH) was present. In N. gonorrhoeaethe CDH was specific for NAD+while the remaining CDH species could utilize either NAD+or NADP+. Only the CDH species within the rRNA Group-II pseudomonad/R. tenuislineage was feedback-inhibited by L-tyrosine, and this correlated with an allosteric pattern where activation of the prephenate dehydratase component of the P-protein by L-tyrosine was relatively poor. However, the CDH enzyme present in N. gonorrhoeaeand A. faecaliswas subject to inhibition by 4-hydroxyphenylpyruvate, this being competitive with respect to the cyclohexadienyl substrate. The monofunctional species of chorismate mutase (CM-F) and cyclohexadienyl dehydratase, widely distributed among the γ-subdivision assemblage and recently shown to be periplasmic enzymes, were demonstrated in Pseudomonas pickettii, a member of rRNA homology Group-II.
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Helicobacter pullorumsp. nov. - genotype and phenotype of a new species isolated from poultry and from human patients with gastroenteritis
Campylobacter-like organisms were isolated from the liver, duodenum and caecum of broiler and layer chickens, and from humans with gastroenteritis. They formed a unique DNA homology group and a polyphasic taxonomic analysis was made of 16 strains. Analysis of the nucleotide sequence of the 16S rRNA gene from seven of the strains identified them as belonging to a single species, within the genus Helicobacter.This conclusion was supported by the studies of relative DNA homology and of total protein electrophoretic patterns. The new species could be biochemically differentiated from other helicobacters and its ultrastructure in the electron microscope was typical of the genus except that the flagellum was not sheathed. We propose the name Helicobacter pullorumsp. nov. for this group. Like H. fennelliaeor H. cinaediit represents another non-gastric urease-negative Helicobacterspecies colonizing the lower bowel. Its isolation from the livers of chickens with vibrionic hepatitis is significant. We describe a species-specific PCR assay for H. pullorumsp. nov. which will facilitate its identification and further studies of its epidemiology.
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Characterization of Thiobacillus caldussp. nov., a moderately thermophilic acidophile
More LessTwo isolates of a novel, moderately thermophilic Thiobacillusspecies have been studied. The isolates, KU and BC13, are Gram-negative, motile bacteria having a pH optimum for growth of 2-2.5 and an optimum growth temperature of 45 °C. Both isolates are capable of chemolithotrophic growth on reduced sulfur substrates. They can also use molecular hydrogen as an electron donor. These two isolates can grow mixotrophically with sulfur or tetrathionate and yeast extract or glucose. The G + C content is 63.1-63.9 mol% and the isolates exhibit no significant DNA homology to any other Thiobacillusspecies. Strains KU and BC13 both contain ubiquinone Q.8. 16S rRNA analysis indicates that these strains belong to a group of bacteria which includes other chemolithotrophic sulfur oxidizers such as T. ferrooxidansand T. thiooxidans. These characteristics distinguish KU and BC13 from any other species described previously and they thus represent the first acidophilic, thermophilic Thiobacillusspecies, named T. caldussp. nov., to be described. The type strain, referred to as strain KU in this paper, has been deposited in the Deutsche Sammlung von Mikroorganismen, Braunschweig, FRG, with the accession number DSM 8584.
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