- Volume 140, Issue 4, 1994
Volume 140, Issue 4, 1994
- Sgm Special Lecture
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- Microbiology Comment
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- Antigens And Immunity
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Reactivity of antibodies to heteroclitic peptides based on the Chlamydia trachomatis major outer-membrane protein
More LessOne problem of peptide vaccines is that antibodies generated against them react poorly with the target sequence on the native protein. Using monoclonal antibodies (mAbs) to the serovar L1 type-specific epitope on the major outer-membrane protein of Chlamydia trachomatis as our model in conjunction with the Pin Technology Epitope Scanning technique, we had previously identified the critical binding site at this epitope as DAVP. Amino acid substitution showed that AV were essential residues for binding. A series of structurally related (heteroclitic) peptides retaining AV were synthesized. Some of these were found to be much more reactive with the model mAb than peptides of cognate sequence. It was hypothesized that the DAVP peptide only approximated to the conformation of the homologous sequence in the native protein, whereas some of the flexible heteroclitic peptides produced conformations which more closely resembled the native constrained sequence. The key question was whether the most reactive heteroclitic peptide would also generate antibody capable of more efficient binding to the native protein. We therefore immunized mice with one of six heteroclitic peptides or one of two native sequence control peptides. The reactivity of these antisera with the peptide immunogens and with native chlamydial elementary bodies was then evaluated by enzyme immunoassay. Pooled antisera to two of the heteroclitic peptides reacted with significantly greater absorbance (P < 0·05) and at higher dilution with whole chlamydiae than did pooled antisera to the control peptides. This suggests that heteroclitic peptides may in some circumstances be useful to increase the reactivity of site-specific antibodies with epitopes on the native protein important for vaccine development or for serodiagnosis.
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- Biochemistry
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Pediocin A, a bacteriocin produced by Pediococcus pentosaceus FBB61
More LessPediocin A, a bacteriocin produced by Pediococcus pentosaceus FBB61, was purified and partially characterized. The purification was achieved by dialysis against PEG 20000, butanol extraction and electroendosmotic preparative electrophoresis. The protocol led to a 7843-fold increase in the specific activity, with 3·9% activity recovered. SDS-PAGE of pediocin A resulted in a single 80 kDa protein band. The antimicrobial compound was sensitive to proteolytic enzymes and heat (10 min at 100 °C). It exhibited inhibition against species of Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Staphylococcus, Enterococcus, Listeria and Clostridium.
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Purification and characterization of a ferulic acid esterase (FAE-III) from Aspergillus niger: specificity for the phenolic moiety and binding to microcrystalline cellulose
More LessAn inducible ferulic acid esterase (FAE-III) has been isolated, purified and partially characterized from Aspergillus niger after growth on oat spelt xylan. The purification procedure utilized ammonium sulphate precipitation, hydrophobic interaction and anion-exchange chromatography. The purified enzyme appeared almost pure by SDS-PAGE, with an apparent M r of 36000. A single band, corresponding to a pl of 3·3 was observed on isoelectric focusing. With methyl ferulate as substrate, the enzyme had a specific activity of 67 IU (mg protein)−1, pH and temperature optima of 5 and 55–60 °C, respectively, and a Km of 2·08 mM and a V max of 175 μmol min−1 (mg protein)−1. The enzyme was also active upon methyl sinapinate, methyl-3,4-dimethoxy cinnamate and methyl p-coumarate, but not benzoic acid methyl esters or methyl caffeate. Similarly, Streptomyces olivochromogenes FAE showed activity against methyl ferulate, methyl sinapinate and methyl p-coumarate, but at a level 420-fold less (on methyl ferulate) than the A. niger esterase. No activity was detected against the benzoate methyl esters. For both enzymes, this shows the necessity for C-3 on the phenol ring to be methoxylated and the aliphatic region of the substrate to be unsaturated. The specific activity of FAE-III on destarched wheat bran was 31 U (mg protein)−1 in the presence of Trichoderma viride xylanase and 3 U (mg protein)−1 in the absence. Apparent pH dependent binding of A. niger FAE-III to microcrystalline cellulose was also demonstrated.
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Formation of 4-hydroxybenzoate in Escherichia coli: characterization of the ubiC gene and its encoded enzyme chorismate pyruvate-lyase
More LessChorismate pyruvate-lyase from Escherichia coli converts chorismate to 4-hydroxybenzoate. The enzyme was enriched 3000-fold by overexpression and chromatographic purification. It has an apparent K m value for chorismate of 6.1 μM and an isoelectric point of pH 6.45. The enzyme activity did not require metal cofactors. Promoter sequences in the 5′ flanking sequences of the ubiCA operon were localized by transcription and translation of active chorismate pyruvate-lyase in vitro from different PCR fragments. Sequencing of the ubiC gene of the mutant strain AN244 revealed a G→A transition resulting in a change from glutamic acid to lysine. A feeding experiment with [1,7-13C2]shikimate confirmed the chorismate pyruvate-lyase as the sole enzymic source of 4-hydroxybenzoate in vivo.
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Morphology and hydrolytic activity of A7, a typing phage of Pseudomonas syringae pv. morsprunorum
More LessBacteriophage A7 has been employed as an indicator for strains of Pseudomonas syringae pv. morsprunorum race 1. Electron microscopy showed that this phage had a hexagonal head, 59 nm in diameter, and a long, flexible, non-contractile tail, 164 nm long and 8 nm wide, containing approximately 15 evenly spaced transverse striations and terminating in a base-plate equipped with six spikes arranged in a radially symmetrical pattern around a central core. Two short fibres projecting at an angle from the base-plate were also visible on some phage particles. Phage A7 can therefore be placed in group B of the classification system of Bradley. Phage particles bound at apparently random sites over the surface of host cells by their base-plates, and after a short time released DNA from their heads. Phage A7 uses lipopolysaccharide (LPS) as its binding site on the bacterial cell surface, removing the d-rhamnan side-chains by the action of a rhamnan hydrolase. The appearance of purified LPS by electron microscopy was either strand-like or vesicular, according to whether it had been stained with phosphotungstate or uranyl acetate respectively. Strands were of approximately uniform width (approx. 9 nm). Vesicular forms included both circles, 25–45 nm in diameter, and larger oval structures of greater electron transparency. The appearance of the LPS did not alter on addition of the phage. Phage particles were observed attached via their base-plates to LPS vesicles, in particular the larger oval vesicles, but were never seen attached to strands. The heads of phage particles attached to LPS were frequently empty. The rhamnanase of phage A7 released the side-chain residues from the LPS as an equimolar mixture of tri- and hexasaccharide. By using 1 h-NMR spectroscopy and methylation analysis, the site of cleavage has been identified as the 2)-α-d-Rhap(1 → 3) residue.
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Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris SK11
More LessAn endopeptidase has been purified from Lactococcus lactis subsp. cremoris SK11. The enzyme is a 70 kDa monomer, strongly inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and phosphoramidon but relatively insensitive to EDTA. It is not significantly inhibited by the thiol enzyme inhibitor p-chloromercuribenzoate nor by the serine protease inhibitor phenylmethylsulphonyl fluoride. The action of the endopeptidase in catalysing the hydrolysis of several peptide hormones has been studied and the hydrolysis products identified by sequence analysis. The enzyme catalyses hydrolysis of peptide bonds in which a hydrophobic amino acid (most commonly a Phe or Leu) residue occupies the position immediately C-terminal to the hydrolysed bond. It thus has a specificity very similar to that of thermolysin. Two of the oligopeptides produced during the early stages of β-casein digestion by the lactococcal cell-wall proteinases were hydrolysed by the endopeptidase, the others were resistant to hydrolysis. Cell fractionation studies have shown that the distribution of endopeptidase activity between the different cell fractions is the same as that of the intracellular marker enzyme fructose bisphosphate aldolase, and thus indicate a cytoplasmic location for the enzyme. These observations argue against a role for this enzyme in the early stages of casein breakdown by the lactococcal proteolytic system.
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Maltose transport in Aeromonas hydrophila: purification, biochemical characterization and partial protein sequence analysis of a periplasmic maltose-binding protein
More LessA clinical isolate of Aeromonas hydrophila was demonstrated to transport [14C]maltose with similar kinetics to enteric bacteria (K m: 0.3μM; V max: 22 nmol min−1 per 109 cells). The uptake of [14C]maltose was completely inhibited in the presence of unlabelled maltose or maltodextrins, whereas other mono- and disaccharides, such as glucose, galactose, sucrose, lactose or melibiose, had no effect. A protein with an apparent molecular mass of 39 kDa (maltose-binding protein; MBP) was identified in osmotic-shock fluid of maltose-grown cells by SDS-gel electrophoresis, and was purified to homogeneity by either amylose affinity chromatography or ion-exchange chromatography. Equilibrium dialysis experiments revealed the ability of the purified protein to bind [14C]maltose with high affinity (KD = 1.6μM). Unlabelled maltose and maltodextrins competed for the binding site. In a reconstitution experiment, A. hydrophila MBP poorly restored the transport activity of a binding-protein-deficient Escherichia coli (ΔmalE) mutant. N-terminal sequence analyses of the purified native protein and of peptides generated by cleavage with CNBr and subsequently separated by HPLC revealed about 56% identical amino acid residues, as compared to enterobacterial MBPs. We conclude that maltose is transported into A. hydrophila via a binding-protein-dependent transport system.
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Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD
More LessTo better understand the reasons for the hyper-accumulation of polyhydroxyalkanoate (PHA) in mutant Azotobacter vinelandii UWD, the kinetic properties of 3-ketothiolase, acetoacetyl-CoA reductase, and β-hydroxybutyrate dehydrogenase were examined. The regulation of the condensation of acetyl-CoA mediated by 3-ketothiolase was normal, in that it was negatively regulated by free CoA, but inhibition was overcome by higher concentrations of acetyl-CoA. Acetoacetyl-CoA from this reaction was reduced to 3-hydroxybutyryl-CoA by an NADPH-specific acetoacetyl-CoA reductase. This enzyme also reduced 3-ketovaleryl-CoA derived from the β-oxidation of C5, C7 or C9 n-alkanoates, but at only 16% of the rate found with the C4-substrate. The acetoacetyl-CoA reductase was determined to be an allosteric enzyme that bound NADPH and acetoacetyl-CoA at multiple binding sites in a general hybrid Ping-Pong random mechanism. The enzyme was negatively regulated by acetoacetyl-CoA, but this was overcome at high concentrations of NADPH. The activity of pyridine nucleotide transhydrogenase was determined to be important for the conversion of NADH in these mutant cells to NADPH and for decreasing the availability of NADP+, which was a negative regulator of the acetoacetyl-CoA reductase. The combination of high acetoacetyl-CoA, the UWD mutation, transhydrogenase activity, and high NADPH appeared to be the conditions promoting PHA formation by strain UWD during active growth on glucose. Degradation of PHA in strain UWD did not appear to be regulated at the level of β-hydroxybutyrate dehydrogenase. This enzyme was unaffected by NADH, was inhibited only 13% by pyruvate and its activity was enhanced by NADPH. The thiolysis of acetoacetyl-CoA also was unusual, in that 3-ketothiolase was not inhibited by acetoacetyl-CoA, but free CoA was a competitive inhibitor in a bireactant Ping-Pong mechanism. This inhibition was overcome by higher concentrations of the normal first substrate, acetoacetyl-CoA. Thus a single thiolase was used for the condensation of acetyl-CoA and the thiolysis of acetoacetyl-CoA, derived from PHA depolymerization or from the β-oxidation of n-alkanoates.
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Ammonia-assimilating enzymes in the basidiomycete fungus Pleurotus ostreatus
More Lessl-Glutamate and l-aspartate are the natural nitrogen sources preferred by the fungus Pleurotus ostreatus. In the vegetative mycelium NAD-dependent glutamate dehydrogenase (NAD-GDH) and glutamine synthetase (GS) were both present, but the highest level of these enzymes was found in the fruiting body. NAD-GDH was derepressed by ammonia and repressed by higher concentrations of l-glutamate. Otherwise, the level of the enzyme in mycelium was independent of the nitrogen source. GS was derepressed by ammonia, l-glutamate, l-aspartate and l-alanine. Repression of GS was observed in the presence of l-glutamine, l-arginine, nitrate, urea and low concentrations of ammonia. An increase in the ratio of glucose to nitrogen resulted in a decrease in the level of GS in the mycelium. The K m values of NAD-GDH for 2-oxoglutarate, ammonia, NADH, l-glutamate and NAD+ were 2·9 mM, 3·3 mM, 11 μM, 0·18 mM and 62 μM, respectively. The K m values of GS for l-glutamate and ammonia were 2·5 and 0·27 mM, respectively. The repression of GS and derepression of NAD-GDH in the presence of low concentrations of ammonia together with glutamate or aspartate could be explained by an inhibition or repression of some amino acid permeases by ammonia. The absence of NADP-dependent glutamate dehydrogenase and glutamate synthase together with the apparent mode of regulation of NAD-GDH and GS synthesis suggest that P. ostreatus assimilates ammonia via GS and NAD-GDH. However, a dissimilatory role of NAD-GDH in the deamination of l-glutamate, due to its very low K m for l-glutamate, is not excluded.
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- Development And Structure
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Isolation and characterization of the outer-membrane proteins of Burkholderia (Pseudomonas) pseudomallei
More LessMembranes obtained from whole-cell lysates of Burkholderia (Pseudomonas) pseudomallei (strain 319a) were separated into four fractions by sucrose density gradient centrifugation. Membranes were characterized by enzymic and chemical analyses, and by SDS-PAGE. Cytoplasmic membranes and two forms of outer membranes (OM-1, OM-2) were detected. The major outer-membrane proteins had M r values of 70000, 38000, 31000, 24000 and 17000. To determine which outer-membrane proteins were common to B. pseudomallei strains, OM-1 fractions from 12 different strains were prepared. SDS-PAGE analysis of these fractions demonstrated that the five major outer-membrane proteins were common to the strains tested. Further studies have shown that an M r 110000 protein, which is oligomeric in that it migrates as an M r 38000 protein upon heating at 95 °C and which is peptidoglycan-associated, serves as a porin in B. pseudomallei. Using proteoliposomes reconstituted from this protein and phospholipid, it was demonstrated by the liposome-swelling assay that this protein acts as a porin through which small saccharides may diffuse. Further characterization of this M r 38000 protein will be important in delineating the role of this molecule in the permeability of the B. pseudomallei outer membrane.
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- Genetics And Molecular Biology
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The glpT and glpQ genes of the glycerol regulon in Bacillus subtilis
More LessThe cloning of the Bacillus subtilis glpT and glpQ genes and their nucleotide sequences are reported. Analysis of mRNA indicates that glpT and glpQ constitute one operon which is transcribed from a sigma A type promoter. The steady state amount of glpTQ mRNA is increased in cells grown in the presence of glycerol 3-phosphate. The 5′ untranslated leader sequence of glpTQ mRNA contains an inverted repeat which shows sequence similarity to repeats present in the leader sequences of glpFK and glpD transcripts. These repeats seem therefore to be essential control elements for all B. subtilis glp genes.
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The hemX gene of the Bacillus subtilis hemAXCDBL operon encodes a membrane protein, negatively affecting the steady-state cellular concentration of HemA (glutamyl-tRNA reductase)
More LessThe Bacillus subtilis hemAXCDBL operon encodes enzymes for the biosynthesis of uroporphyrinogen III from glutamyl-tRNA. The function of the hemX gene product was studied in this work. The deduced amino acid sequence suggests HemX to be an integral 32 kDa membrane protein. This was confirmed by experiments using Escherichia coli minicells and hemX-phoA gene fusions. Deletion of the hemX gene from the Bacillus subtilis chromosome demonstrated that this gene is not required for haem synthesis. However, the deletion strain was found to overexpress the hemA gene product, glutamyl-tRNA reductase. A combination of results obtained with B. subtilis hemA and hemX in Escherichia coli and Bacillus subtilis shows that HemX negatively affects the steady-state cellular concentration of HemA protein. The mechanism by which HemX affects the HemA concentration is unclear.
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Molecular cloning and sequence analysis of yps-3, a yeast-phase-specific gene in the dimorphic fungal pathogen Histoplasma capsulatum
More LessGenes specifically expressed in the parasitic yeast phase of Histoplasma capsulatum have been cloned to clarify the mechanisms underlying both pathogenesis and morphogenesis in this human dimorphic fungal pathogen. Previous studies have determined that the yeast-phase-specific gene, yps-3, is expressed differentially in two non-isogenic strains which differ in their thermotolerance and virulence. We have cloned the yps-3 homologues from the high virulence (G217B) and low virulence (Downs) strains, and obtained a partial cDNA clone representing the expressed gene from H. capsulatum G217B. The Downs clone harbours a 287 bp insertion sequence that disrupts a long ORF defined by the yps-3 G217B cDNA. Although the insertion sequence contains features reminiscent of mobile genetic elements, including 15 bp direct repeats of flanking sequence, it is not randomly distributed in the H. capsulatum genome. S1 nuclease analysis was utilized to map the 5′ end of the expressed yps-3 gene in G217B to potential regulatory regions which are largely homologous in both strains. This finding may point to a deficiency in a temperature inducible regulatory protein in the low virulence, temperature-sensitive Downs strain. The nucleotide sequence of the yps-3 gene and the predicted amino acid sequence of its product represents the first report of phase-specific gene and protein sequences in this widely distributed fungal pathogen. Further analysis of the product encoded by the yps-3 gene may provide significant insight into the pathogenic repertoire of H. capsulatum.
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Regulation of gene expression by the HlyX protein of Actinobacillus pleuropneumoniae
More LessThe hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae and the fnr gene of Escherichia coli encode very similar proteins. The hlyX gene is able to complement Δfnr mutations and will permit the growth of E. coli fnr strains in nitrate minimal salts medium under anoxic conditions. In addition, the hlyX gene product appears to induce the expression of a latent haemolytic activity as evidenced by the presence of a strong zone of haemolysis around E. coli (hlyX +) colonies grown on bovine or ovine blood. In this study, the ability of the hlyX gene product to induce haemolytic activity and regulate expression of frdA and its own gene was examined in the presence of various carbon sources and in the presence and absence of iron; fnr was included for comparison. The HlyX protein was able to induce the synthesis of the latent E. coli haemolytic activity only under anoxic conditions. Haemolytic activity was highest during the late exponential phase and then levelled off in the stationary phase. The hlyX gene product was able to activate the expression of a ø (frdA’-lacZ) in E. coli JRG1787 (Δfnr); however, the level of expression depended on carbon source, growth phase and copy number. Like fnr, the hlyX gene product appeared to affect its own synthesis but the nature and extent of regulation depended not only on the presence of oxygen but also on growth conditions.
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Aerobic expression of the cyf gene encoding cytochrome c-553 from Desulfovibrio vulgaris Hildenborough in Escherichia coli
More LessMonohaem cytochrome c-553 from Desulfovibrio vulgaris Hildenborough is encoded by the cyf gene which could be expressed in Escherichia coli to yield the periplasmic holoform of cytochrome c-553. Covalent haem attachment was shown by labelling with 5-amino[4-14C]laevulinic acid, the immediate precursor for haem biosynthesis. Visible-absorption spectroscopy demonstrated that the haem environment in the recombinant protein did not differ from the native protein. Optimal expression was obtained under aerobic conditions. Furthermore, efficient insertion of haem into a cytochrome c-553-β-lactamase fusion protein could be demonstrated. We suggest that the varying success in heterologous expression of c-type cytochromes in E. coli may arise as a result of differences in the physical properties of the apoproteins.
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Cloning and sequencing of the Proteus mirabilis gene for a single-stranded DNA-binding protein (SSB) and complementation of Escherichia coli ssb point and deletion mutations
More LessThe gene of Proteus mirabilis coding for a single-stranded DNA-binding protein (SSB) was cloned in Escherichia coli from a genomic library. It restored the UV resistance and the rate of cell division of an E. coli ssb-113 mutant to the same extent as the cloned E. coli ssb gene did. An E. coli mutant with deleted ssb was viable with the P. mirabilis ssb + gene provided on a single-copy-number plasmid and had the same cell division rate as with the E. coli ssb + gene on the same vector plasmid. The recovery from UV damage of an excision repair deficient (uvrA) mutant deleted for the ssb gene was identical with the ssb + gene from P. mirabilis or E. coli, suggesting full substitution in recombinational DNA repair of the homologous by the heterologous SSB protein. The nucleotide sequence of the gene revealed that the SSB has 81% amino acid sequence homology with the E. coli SSB and only 58–63% with various plasmid SSBs. The data provide evidence that the bacterial chromosomally coded SSBs and the plasmid encoded SSBs constitute separate groups.
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Transposon-mediated mobilization of chromosomally located catabolic operons of the CAM plasmid by TOL plasmid transposon Tn4652 and CAM plasmid transposon Tn3614
More LessThe CAM (camphor degradation) plasmid is integrated into the chromosome of Pseudomonas putida PaW-line strains and is not selftransferable as a plasmid via conjugation. Our results show that the mobilization of chromosomally located CAM and the integration of cam-operons into the chromosome of the new Cam+ transconjugants is a recA-independent process mediated by transposons Tn4652 (17 kbp) and Tn3614 (7.2 kbp). Transposon Tn3614 is apparently identical to the left-hand and the right-hand sequences of the TOL plasmid pWWO transposon Tn4654. The insertion of Tn401 inside the left-hand terminal IR of Tn4652 completely inhibited the mobilization of CAM. According to our data transposons Tn4652 and Tn3614 together with CAM plasmid catabolic operons are integrated into the chromosome. We propose that in eseudomonads the transposons Tn4652 and Tn3614 play a key role in the evolution and spread of new catabolic plasmids in nature.
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Three tuf-like genes in the kirromycin producer Streptomyces ramocissimus
We have identified, cloned and sequenced three tuf-like genes from Streptomyces ramocissimus (Sr.), the producer of the antibiotic kirromycin which inhibits protein synthesis by binding the polypeptide chain elongation factor EF-Tu. The tuf-1 gene encodes a protein with 71 % amino acid residues identical to the well characterized elongation factor Tu of Escherichia coli (Ec.EF-Tu). The genetic location of tuf-1 downstream of a fus homologue and the in vitro activity of Sr. EF-Tu1 show that tuf-1 encodes a genuine EF-Tu. The putative Sr. EF-Tu2 and Sr.EF-Tu3 proteins are 69% and 63% identical to Ec.EF-Tu. Homologues of tuf-1 and tuf-3 were detected in all five Streptomyces strains investigated, but tuf-2 was found in S. ramocissimus only. The three tuf genes were expressed in E. coli and used to produce polyclonal antibodies. Western blot analysis showed that Sr.EF-Tu1 was present at all times under kirromycin production conditions in submerged and surface-grown cultures of S. ramocissimus and in germinating spores. The expression of tuf-2 and tuf-3 was, however, below the detection level. Surprisingly, Sr.EF-Tu1 was kirromycin sensitive, which excludes the possibility that EF-Tu is involved in the kirromycin resistance of S. ramocissimus.
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- Pathogenicity And Medical Microbiology
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Chemical and immunological analysis of the Aspergillus fumigatus cell wall
More LessHyphal-wall preparations of Aspergillus fumigatus have been analysed by sequential treatment with KOH, nitrous acid and again with KOH. By acidification of the alkali-soluble extract, a polyglucose was precipitated which showed an X-ray diffraction pattern similar to that of (1·3)-α-glucan. The remainder of the alkali-soluble fraction was precipitated with ethanol; it contained all the mannose, galactose and protein of the wall and, in addition, 6·2% of the amino sugars. This wall-associated glycoprotein, following SDS-PAGE and immunoblotting, reacted with antisera raised against several mycelial extracts of A. fumigatus. Sera from patients with aspergilloma have antibodies which recognize components of this glycoprotein. The glycoprotein nature of these antigens was shown by their ability to bind Lens culinaris lectin. In addition, the antigen/antibody binding could be disrupted by exposure of antigen to periodate oxidation, hydrolysis with dilute acid or pretreatment with a large excess of an exo-β-d-galactofuranosidase. The alkaliinsoluble fraction consisted of a covalently linked glucan-chitin complex. Nitrous acid treatment, which specifically disrupts glycosidic linkages involving glucosamine, did not solubilize much material but changed the X-ray diffraction pattern from diffuse to a pattern showing the characteristic lines of crystalline (1·3)-β-glucan and chitin. Most of the glucan became alkali-soluble after this treatment, and the insoluble residue appeared to contain crystalline chitin.
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Variation in outer-membrane protein and lipopolysaccharide profiles of Pasteurella haemolytica isolates of serotypes A1 and A2 obtained from pneumonic and healthy cattle
More LessThe outer-membrane protein (OMP) and lipopolysaccharide (LPS) profiles of 29 isolates of Pasteurella haemolytica serotypes A1 (18 isolates) and A2 (11 isolates), obtained from pneumonic (13 isolates) or healthy (16 isolates) cattle, were compared by SDS-PAGE and Western blot analysis. Coomassie-blue-stained OMP profiles of serotype A1 and A2 isolates could be distinguished from each other by differences in both major and minor proteins. Whereas the OMP profiles of the serotype A1 isolates were extremely uniform in stained gels, there was variation in the mobilities of high-molecular-mass minor proteins and one of the major proteins of serotype A2 isolates. Differences in the OMP profiles of isolates within both the A1 and A2 serotypes were more clearly distinguished by Western blotting than by staining after SDS-PAGE. Thus, by Western blot analysis, four distinct OMP profiles were identified within the serotype A1 and A2 isolates, respectively. The profiles of the serotype A1 isolates were designated OMP types 1.1, 1.2, 1.3 and 1.4; those of the serotype A2 isolates were designated OMP types 2.1, 2.2, 2.3 and 2.4. Three distinct LPS profiles were recognized among the isolates which, by comparison with previously described LPS types, were identified as smooth LPS type 1 and rough LPS types 3 and 5. Isolates of serotype A1 consisted of LPS type 1 only, whereas isolates of serotype A2 consisted of LPS types 3 or 5. OMP and LPS analysis of P. haemolytica has applications in epidemiological and virulence studies.
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Phospholipids of Mycobacterium intracellular inhibit T cell blastogenesis
More LessA crude lipid fraction obtained from Mycobacterium intracellulars (MI whole lipids) suppressed concanavalin A (Con A)-induced blastogenesis of murine spleen cells (SPCs). Among three lipid fractions, the phospholipid fraction possessed the highest inhibitory activity, followed by the polar mycoside fraction, but the apolar mycoside fraction showed no activity. Since MI whole lipid and phospholipid fractions inhibited the Con A-induced proliferative response of SPCs which had been treated with Con A before the addition of the lipid fractions, their action is not due to hindrance of Con A binding to T cells. These two MI lipid fractions reduced IL-2-producing ability, acquisition of IL-2 reactivity, and expression of IL-2 receptors in Con A-stimulated T cells. However, they did not affect IL-2-induced proliferation of an IL-2-dependent cytotoxic T cell line, CTLL-2. When SPCs were pretreated with either MI whole lipid or phospholipid fraction for 24 h, an irreversible reduction in Con A responsiveness was seen only in the phospholipid-treated SPCs. The action of these MI lipid fractions was phase-dependent and they exhibited considerably decreased but still significant inhibitory activity against Con A blastogenesis of SPCs, even when they were added at 24 h after the initiation of SPC culture with Con A. MI whole lipids and the three lipid fractions (polar mycoside, apolar mycoside, and phospholipid fractions) did not exhibit suppressor cellinducing activity, while MI whole lipid fraction antagonized the Con A-mediated generation of suppressor cells. Silica gel thin layer chromatography of the phospholipid fraction showed four spots containing phosphate and one spot without. SPC Con A blastogenesis-inhibitory activity was shared by the two least polar phosphate-containing substances. One of those apparently contained amino groups and carbohydrate moieties. The second component contained no such moieties.
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The effect of growth rate and haemin on the virulence and proteolytic activity of Porphyromonas gingivalis W50
More LessPorphyromonas gingivalis strain W50 was grown under haemin-limitation and haemin-excess conditions in a chemostat at pH 7.5. The maximum specific growth rate (μmax) was determined at both haemin concentrations (μmax = 0.236 ± 0.052 and 0.271 ± 0.039 h-1 respectively). This enabled dilution rates to be adjusted so that the virulence and enzyme activity of haemin-limited and haemin-replete cells could be compared at identical relative growth rates (μrel) of 0.25, 0.50 and 0.75 of their respective μmax. The data showed that the fastest growing cells were significantly more virulent than those grown more slowly, irrespective of haemin concentration. However, at each growth rate tested, cells grown under haemin-excess conditions were always more virulent than haemin-limited cells. Trypsin-like enzyme activity of whole cultures was also greater at each growth rate under haemin-excess conditions while, conversely, collagenolytic activity was generally higher in haemin-limited cultures. Thus, although growth rate had an effect on the virulence and enzyme activity of P. gingivalis, the availability of haemin for growth was the most significant factor.
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Molecules of Streptococcus gordonii that bind to Porphyromonas gingivalis
More LessInterbacterial binding is considered an important colonization mechanism for many of the organisms that inhabit dental plaque. Porphyromonas gingivalis, a periodontal pathogen, can adhere to species that comprise early plaque, such as Streptococcus gordonii. In this study, the molecules of S. gordonii G9B that mediate binding to P. gingivalis were investigated. Biotinylated surface molecules of S. gordonii were extracted and mixed with P. gingivalis cells. Interactive streptococcal components were identified by SDS-PAGE of the P. gingivalis cells followed by electroblotting, and visualization of the adsorbed streptococcal molecules with streptavidin-alkaline phosphatase. S. gordonii molecules of 45 kDa and a doublet of 62/60 kDa were observed to bind to P. gingivalis. Polyclonal antibodies raised to the 62/60 kDa proteins inhibited the binding interaction. These antibodies demonstrated an antigenic relationship between the 62/60 kDa molecules and the 45 kDa protein. Both molecules were also antigenically related to, and may be breakdown products of, a larger molecule of 170 kDa which is antigenically related to the P1 antigen of S. mutans. Cloning and expression in Enterococcus faecalis of the gene for the P1-like molecule from S. gordonii M5 resulted in a phenotype that expressed the 62/60 kDa and 45 kDa antigens and was capable of binding to P. gingivalis. These results suggest that a P1-like molecule in S. gordonii is involved in adherence to P. gingivalis. Processing of the P1-like molecule into smaller fragments of 62/60 kDa and 45 kDa may be required for binding activity.
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Hydrolytic enzymes and lectin-binding activity of black-pigmented anaerobic rods
More LessRecent taxonomic studies on black-pigmented anaerobic rods, a group of bacteria found on mucosal surfaces of humans and animals, led to the subdivision of existing species and to the creation of new species. The aim of this study was to characterize all 11 currently recognized species of black-pigmented bacteria (55 strains) for their ability to hydrolyse a variety of natural and synthetic substrates and for their lectin reactivity. Although most of the strains demonstrated some activity against proteinaceous substrates, Porphyromonas gingivalis was the only species able to hydrolyse type I collagen. Most strains possessed glycylprolyl protease activity, elastase-like activity and phospholipase C activity, whereas trypsin-like activity was restricted to P. gingivalis, Porphyromonas salivosa and Bacteroides macacae. β-Lactamase activity was demonstrated in five strains belonging to the saccharolytic group. The lectin reactivity of the bacteria was determined by a dot-blot procedure using horseradish-peroxidase-conjugated lectins. Three lectins, LOTUS A, RCA-1 and ConA, failed to react with any of the bacteria tested. WGA reacted strongly with the cell surface of human biotypes of asaccharolytic black-pigmented bacteria (P. gingivalis, Porphyromonas asaccharolytica and Porphyromonas endodontalis) and Prevotella intermedia. The animal biotype strains of P. gingivalis showed a higher affinity for SBA and PNA than for WGA.
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- Physiology And Growth
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Effects of oxygen limitation on sugar metabolism in yeasts: a continuous-culture study of the Kluyver effect
More LessGrowth and metabolite formation were studied in oxygen-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 growing on glucose or maltose at a dilution rate of 0·1 h−1 With either glucose or maltose S. cerevisiae could be grown under dual limitation of oxygen and sugar. Respiration and alcoholic fermentation occurred simultaneously and the catabolite fluxes through these processes were dependent on the magnitude of the oxygen feed. C. utilis could also be grown under dual limitation of glucose and oxygen. However, at very low oxygen feed rates (i.e. below 4 mmol l−1 h−1) growth was limited by oxygen only, as indicated by the high residual glucose concentration in the culture. In contrast to S. cerevisiae, C. utilis could not be grown anaerobically at a dilution rate of 0·1 h−1. With C. utilis absence of oxygen resulted in wash-out, despite the presence of ergosterol and Tween-80 in the growth medium. The behaviour of C. utilis with respect to maltose utilization in oxygen-limited cultures was remarkable: alcoholic fermentation did not occur and the amount of maltose metabolized was dependent on the oxygen supply. Oxygen-limited cultures of C. utilis growing on maltose always contained high residual sugar concentrations. These observations throw new light on the so-called Kluyver effect. Apparently, maltose is a non-fermentable sugar for C. utilis CBS 621, despite the fact that it can serve as a substrate for growth of this facultatively fermentative yeast. This is not due to the absence of key enzymes of alcoholic fermentation. Pyruvate decarboxylase and alcohol dehydrogenase were present at high levels in maltose-utilizing cells of C. utilis grown under oxygen limitation. It is concluded that the Kluyver effect, in C. utilis growing on maltose, results from a regulatory mechanism that prevents the sugar from being fermented. Oxygen is not a key factor in this phenomenon since under oxygen limitation alcoholic fermentation of maltose was not triggered.
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Propionate metabolism in Saccharomyces cerevisiae: implications for the metabolon hypothesis
Aerobic, glucose-limited chemostat of Saccharomyces cerevisiae CBS 8066 cometabolized propionate when this compound was added to the reservoir medium. Co-metabolism of propionate led to an increase of the biomass and protein yields. Attempts to grow S. cerevisiae on propionate as a sole source of carbon and energy were not successful. Activities of propionyl-CoA synthetase in cell-free extracts were sufficient to account for the rates of propionate consumption observed in the chemostat cultures. Activities of propionyl-CoA carboxylase, a key enzyme of the methylmalonyl-CoA pathway of propionate metabolism, were negligible. In contrast, activities of 2-methylcitrate synthase, a key enzyme activity of the 2-methylcitrate pathway of propionate metabolism, increased substantially with increasing propionateto-glucose ratios in the reservoir media, and were sufficient to account for the propionate consumption rates observed in the chemostat cultures. This suggested that the 2-methylcitrate pathway is the major pathway of propionate metabolism in S. cerevisiae. In the literature, labelling patterns observed after incubation of this yeast with [3-13C]propionate have been interpreted as evidence for channelling of tricarboxylic acid (TCA) cycle intermediates, possibly as a consequence of the organization of TCA cycle enzymes in a metabolon. However, this interpretation of 13C-labelling patterns rested on the assumption that propionate metabolism in S. cerevisiae occurs via the methylmalonyl-CoA pathway. Since the distribution of 13C in alanine reported in the literature is fully compatible with a major role of the 2-methylcitrate pathway in propionate metabolism, it cannot be interpreted as evidence for the existence of a TCA cycle metabolon in S. cerevisiae.
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Analysis of the induction of general stress proteins of Bacillus subtilis
In Bacillus subtilis stress proteins are induced in response to different environmental conditions such as heat shock, salt stress, glucose and oxygen limitation or oxidative stress. These stress proteins have been previously grouped into general stress proteins (Gsps) and heat-specific stress proteins (Hsps). In this investigation the N-terminal sequences of 13 stress proteins of B. subtilis were determined. The quantification of the mRNA and the analysis of the protein synthesis pattern support the initial hypothesis that the chaperones DnaK and GroEL are Hsps in B. subtilis. In contrast, the recently described proteins GsiB, Ctc and RsbW belong to a class of Gsps that are induced by various stresses including heat shock. The main part of the Gsps described in this study failed to be induced in the sigB deletion mutant ML6 in response to heat shock. However, all the five Hsps were induced in this mutant in response to heat shock. These data indicate that SigB plays a crucial role in the induction of general stress genes, but is dispensable for the induction of Hsps.
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Regulation of xylanolytic enzymes in Bacillus subtilis
More LessThe synthesis of the xylanolytic enzymes β-xylanase and β-xylosidase of Bacillus subtilis was studied. In contrast to many catabolic extracellular enzymes, β-xylanase was synthesized constitutively during exponential growth and was not repressed by glucose. β-Xylosidase synthesis was induced 100-fold by xylose and repressed 100-fold by glucose. Carbon catabolite repression was abolished in a ccpA mutant. Titration experiments using a multicopy operator sequence respondible for carbon catabolite repression indicated that the gene encoding β-xylosidase is part of the same carbon catabolite repression regulon as the amyE and bgIS genes.
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Importance of stored triacylglycerols in Streptomyces: possible carbon source for antibiotics
More LessSubmerged cultures of four Streptomyces species accumulated triacylglycerol (TAG), ranging from 50 to 150 mg (I medium)−1, during the post-exponential phase of growth. S. lividans also produced glycogen (80 mg I−1). Identity of TAG species was confirmed after TLC, by mass spectroscopy and by quantitative IR spectroscopy and reaction of the hydroxamate derivatives with ferric chloride. This is the first substantive report showing the storage of TAG in bacteria. Distribution of diacylglycerol acyltransferase (TAG synthase) activity from S. coelicolor and S. lividans during incubation on different media paralleled the formation of TAG. Accumulation of TAG may be necessary to maintain cell integrity after glucose becomes exhausted from the medium, and also to provide the C2 units needed for subsequent biosynthesis of acetate-derived antibiotics in appropriate species. Actinorhodin was formed by S. coelicolor when grown on YEME medium only after exhaustion of glucose; the carbon source may therefore originate from TAG. All the organisms examined in this study formed isoprenoid-derived hydrocarbons (up to 3 mg I−1) which were identified as squalene plus hydrogenated derivatives.
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Nitrogen fixation by Plectonema boryanum has a photosystem II independent component
More LessThe non-heterocystous filamentous cyanobacterium Plectonema boryanum showed a several-fold decline in photosystem II dependent O2 evolution during the diazotrophic phase of growth. A sharp fall in the amounts of light-harvesting pigments and an uncoupling between the electron transfer from the photosystem II complex to the quinone acceptors may lead to the depression of light-dependent O2 evolution during the diazotrophic phase. Nitrogen fixation as well as CO2 fixation required light, but were partly supported independently of photosystem II. A stimulation of photosystem I and a depression of photosystem II occurred during the diazotrophic phase of a nitrogen-fixing culture of P. boryanum.
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- Plant-Microbe Interactions
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Characterization of cell surface components of Azospirillum brasilense Sp7 as antigenic determinants for strain-specific monoclonal antibodies
Monoclonal antibodies (mAbs) with high specificity for Azospirillum brasilense Sp7, and which bind to different antigenic determinants, were characterized using Western blot techniques applied to one- and two-dimensional fingerprints of the outer-membrane components, and by immunogold labelling combined with transmission electron microscopy. One class of mAbs, which bound to A. brasilense Sp7 and the closely related strain Cd, recognized a 100 kDa protein subunit of the polar flagellum. Two classes of strain-specific mAbs for A. brasilense Sp7 bound, respectively, to a 85 kDa outer-membrane protein and to polysaccharide.
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- Systematics
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Intraspecific molecular variation among Trichoderma harzianum isolates colonizing mushroom compost in the British Isles
More LessThe genetic diversity in Trichoderma harzianum isolates from mushroom compost was assessed using various molecular techniques. Restriction fragment length polymorphism (RFLP) analysis of ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) divided the 81 isolates into three major groups, 1, 2 and 3. There was no variation within a group in rDNA, while a low degree of polymorphism was detected in mtDNA. Random amplified polymorphic DNA (RAPD) analysis of 30 randomly chosen isolates, with six primers, in general confirmed the RFLP groups. Nucleotide sequence determination of rDNA internal transcribed spacer (ITS) 1 revealed three distinct ITS types, 1, 2 and 3, possessed by isolates from the respective groups 1, 2 and 3. Based on these molecular data, group 2 isolates, which are aggressive colonizers of mushroom compost, could be clearly distinguished from the isolates belonging to the other two groups.
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Two types of 16S rRNA gene are found in Campylobacter helveticus: analysis, applications and characterization of the intervening sequence found in some strains
In the recently described species Campylobacter helveticus, two sizes of PCR amplicon were detected with primers homologous to conserved regions of the 16S rRNA gene. A conventionally sized gene was sequenced from the type strain, NCTC 12470, placing the new species as phylogenetically related to C. upsaliensis and the thermotolerant campylobacters. This nucleotide sequence enabled PCR primers to be designed for use in rapid molecular identification of C. helveticus and its closest phylogenetic relative, C. upsaliensis. When this assay was employed to characterize 22 ‘C. upsaliensis-like’ isolates, twelve were identified as C. helveticus and nine as C. upsaliensis, in agreement with data obtained with a C. helveticus-specific DNA probe. A 550 bp amplicon internal to the 16S rRNA gene of C. helveticus was used to determine restriction fragment length polymorphisms (RFLPs) in genomic Southern blots, confirming that the copy number of the C. helveticus gene was three, and identifying nine 16S rRNA gene profiles. In 5/12 C. helveticus isolates identified by PCR, an enlarged amplicon was detected. The enlarged 16S rRNA gene of one of these strains, NCTC 12838, was sequenced and shown to contain an atypical intervening sequence (IVS) of 148 nucleotides. The position and size of such an IVS was inferred in the other four isolates by PCR with primers 5′ and 3′ to its position in NCTC 12838. This is a first report of an IVS in the 16S rRNA gene of a eubacterium.
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Rhodobacter marinus sp. nov.: a new marine hydrogen producing photosynthetic bacterium which is sensitive to oxygen and sulphide
More LessA new non sulphur photosynthetic bacterium, strain NKPB 0021, was isolated from sea water samples and assigned to the genus Rhodobacter on the basis of morphology and presence of vesicular photosynthetic membranes. Cells are motile rods, 0.6–1.2 μm in diameter and multiply by binary fission. This isolate is unusual since it is oxygen sensitive and cannot grow aerobically. Strain NKPB 0021 grows best anaerobically in the light and requires NaCl for growth (optimum 1.5%). Cells are capable of assimilatory sulphate reduction, and are sensitive to sulphide, with no growth above 0.7 mM sulphide. The mean DNA base composition is 66.7 mol% G + C. On the basis of this study, we propose strain NKPB 0021 to be the type strain of a new species, Rhodobacter marinus.
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- Genome Analysis
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Cloning the ribosomal RNA operons of Mycoplasma flocculare and comparison with those of Mycoplasma hyopneumoniae
More LessIn contrast to other mycoplasma species the 16S/23S rRNA and 5S rRNA operons of Mycoplasma flocculare and Mycoplasma hyopneumoniae map at least 150 kb apart (20% of the genome). Both operons from M. flocculare have been cloned and sequenced. The 23S rRNA gene sequence showed 96.7% homology with the corresponding gene of M. hyopneumoniae, equalling that found earlier for 16S rRNA and confirming the close phylogenetic relationships of these organisms. A possible upstream promoter was identified. Sequence elements upstream and downstream from each structural gene could form a stem needed for maturation of the immature rRNA transcript to mature 16S and 23S rRNA. We also identified two possible stem-and-loop sequences 3' to the 23S rRNA gene. The 5S rRNA gene itself also showed high homology with the corresponding structural gene of M. hyopneumoniae, although the upstream and downstream sequences were highly heterologous.
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- Corrigendum
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