- Volume 135, Issue 12, 1989
Volume 135, Issue 12, 1989
- Biochemistry
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Properties of a Phosphocarrier Protein (HPr) Extracted from Intact Cells of Streptococcus sanguis
More LessCells of Streptococcus sanguis strain Challis were incubated with sodium lauroylsarcosinate to extract surface proteins. A polypeptide of apparent molecular mass 16 kDa comprising about 12% of the extract was purified using anion-exchange chromatography. The polypeptide was shown to be a phosphocarrier protein (HPr) that could also be found in the soluble (cytoplasmic) fraction from cells broken by homogenization with glass beads. In vivo labelling of S. sanguis cells with 32Pi showed that the polypeptide carried a heat- and acid-stable phosphorylation and that during sucrose starvation the HPr became dephosphorylated. Antiserum raised to the S. sanguis HPr reacted on Western blots with HPrs from all oral streptococci tested, together with strains of S. pyogenes and S. salivarius, but not with HPrs from S. faecalis or S. bovis, nor with proteins from Staphylococcus aureus, Bacillus subtilis, Actinomyces viscosus and various lactobacilli. The 5. sanguis HPr had a high content of alanine (17·2%) and was similar in overall amino acid composition to the HPrs from S. mutans and S. salivarius. The N-terminal residues (to 37) of the S. sanguis HPr showed strong sequence identity (82%) with the N-terminal sequence of S. faecalis HPr. It is suggested that HPr in S. sanguis is associated closely with the cytoplasmic membrane. Non-disruptive methods of removing cell-surface proteins from streptococci effect release of HPr and possibly other cytoplasmic components.
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Purification and Partial Characterization of a Major Outer-membrane Protein of Fusobacterium nucleatum
More LessThe major outer-membrane proteins of 40-41 kDa were identified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in Fusobacterium nucleatum strains ATCC 10953, ATCC 25586, F3, F6 and Fevl. The proteins were purified by preparative gel electrophoresis. Their behaviour in gel filtration and gel electrophoresis, their sensitivity to proteolytic enzymes, and their amino acid composition were investigated. The purified proteins were partly sequenced from the N-terminal end. A 36·5 kDa portion was protected against extrinsic proteolytic (trypsin, chymotrypsin or pronase) digestion of whole cells. This polypeptide was isolated and partially sequenced from the N-terminal end. From these data and data from extrinsic iodination it was concluded that the N-terminal end of the protein is probably exposed on the surface of the cell. A database search revealed amino acid sequence similarity in an Ala-Pro-rich region of outer-membrane protein A (OmpA) in other Gram-negative bacteria.
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Inducible Ribosomal RNA Methylation in Streptomyces lividans, Conferring Resistance to Lincomycin
More LessStreptomyces lividans TK21 possesses inducible ribosomal RNA methylase activity that confers high-level resistance to lincomycin and lower levels of resistance to certain macrolides. The methylase gene (designated lrm) is inducible by erythromycin and other macrolides and also by celesticetin (a lincosamide) but not by lincomycin. The lrm enzyme monomethylates the N 6-amino group of adenosine at position 2058 within 23S-like ribosomal RNA.
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Purification and Properties of NADP-dependent Glutamate Dehydrogenase from Streptomyces fradiae
Streptomyces fradiae has two chromatographically distinct forms of glutamate dehydrogenase (GDH): one GDH utilizes NAD as coenzyme, the other uses NADP. The intracellular level of both GDHs is strongly regulated by the nitrogen source in the growth medium. NADP-dependent GDH was purified to homogeneity from crude extracts of S. fradiae. The M r of the native enzyme was determined to be 200000 by size-exclusion high-performance liquid chromatography whereas after sodium dodecyl sulphate-polyacrylamide gel electrophoresis one major band of M r 49000 was found, suggesting that the enzyme is a tetramer. The enzyme was highly specific for the substrates 2-oxoglutarate and l-glutamate, and required NADP, which could not be replaced by NAD, as a cofactor. The pH optimum was 9·2 for oxidative deamination of glutamate and 8·4 for reductive amination of 2-oxoglutarate. The Michaelis constants (K m) were 28·6 mm for l-glutamate and 0·12 mm for NADP. K m values for reductive amination were 1·54 mm for 2-oxoglutarate, 0·07 mm for NADPH and 30·8 mm for NH4 +. The enzyme activity was significantly reduced by adenine nucleotides, particularly ATP.
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Purification of a Ca2+/Calmodulin-dependent Protein Kinase from Baker's Yeast
More LessA Ca2+- and calmodulin-dependent protein kinase was purified from baker’s yeast to near homogeneity. At pH 7·5 and 0·1 m-NaCl it has a native molecular mass close to 100 kDa, and is a dimer of apparently identical 56 kDa autophosphorylatable subunits. At 60 μm-CaCl2 and with mixed histones as substrate, half-maximal activation required concentrations of beef calmodulin above 1 μm. At 0·14 μm-beef calmodulin the enzyme showed apparent negative cooperativity towards ATP, with limiting apparent K m values of 4 μm and 60 μm ATP. The enzyme has a broad substrate specificity in vitro, including two yeast proteins that yield, respectively, 50 kDa and 200 kDa phosphopolypeptides.
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Escherichia coli Molybdoenzymes Can Be Activated by Protein FA from Several Gram-negative Bacteria
More LessSix Gram-negative bacteria (Klebsiella pneumoniae, Erwinia chrysanthemi, Proteus vulgaris, Serratia marescens, Salmonella typhimurium, and Pseudomonas aeruginosa) were shown to contain an FA-type protein capable of activating aponitrate reductase, apotrimethylamine N-oxide reductase and apoformate dehydrogenase of Escherichia coli. Protein FA activity was highest in Erwinia chrysanthemi and lowest in Pseudomonas aeruginosa. All the species also contained the low-M r (≤ 1500) heat-resistant material previously reported to be necessary for the protein-FA-dependent activation of E. coli chlB nitrate reductase.
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- Ecology
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Enhancement of Bacteriophage φX-174 Plaques by Homoionic Clay Minerals
More LessThe incorporation of selected particulates or charged polymers into qualitative and quantitative assay systems has been found to increase virus specific infectivity. The purpose of this study was to identify a mechanism(s) to explain the effect of charged particulates upon plaquing efficiency. The clay minerals kaolinite (K), montmorillonite (M), with the bacteriophage øX-174 and its host Escherichia coli, were used as a model system. Enhanced bacteriophage infectivity (expressed as plaque-forming units) occurred at K and M concentrations in the agar overlay of 0·14–0·0 and 0·01·15 mg ml−, respectively. A maximum increase of bacteriophage titre (to 180–230% of the control value) occurred at 0·18–0·20 and 0·06–0·08 mg ml−K and M, respectively. Increased infectivity was related to the type of clay mineral and to the cation saturating the exchange complex in the order K homoionic (i.e. saturated with a single cation type) to Na+> Mg2+> K+and M homoionic to Mg2+> Na+> K+. Enhanced infectivity of the bacteriophage was not related to the surface area of K nor to the external or total surface area of M. The increase of bacteriophage titre above that of the control was only minimally related to cation-exchange capacity, but was associated with the anion-exchange capacity of the clay. The increased plaquing efficiency is ascribed to the formation of high-density bacteria-clay microcosms which, in turn, modify bacteriophage contact and adsorption to the host cell.
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- Genetics And Molecular Biology
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Molecular Analysis of a Novel Glutamine Synthetase of the Anaerobe Bacteroides fragilis
More LessThe nucleotide sequence of a 2777 bp DNA segment containing the Bacteroides fragilis glnA gene was determined. The B. fragilis glnA open reading frame of 2187 bp encoded a glutamine synthetase (GS) subunit of 729 amino acid residues with a calculated M r of 82827. The apparent M r of the GS subunit determined by SDS-PAGE was approximately 75000. A single mRNA transcription start point was identified upstream of the B. fragilis glnA open reading frame. The B. fragilis GS subunit is approximately 270 and 400 amino acids longer than the GSI and GSII subunits, respectively, of other prokaryotes and eukaryotes. The GSI and GSII holoenzymes are dodecamers and octamers respectively, whereas the GS of B. fragilis is a hexamer. Although GSI and GSII subunits show amino acid similarity in five conserved regions, this similarity is not strongly conserved in the B. fragilis GS. The GS of B. fragilis is not regulated by adenylylation and lacks the adenylylation site. It also lacks the Trp residue associated with the active site in GSI and GSII enzymes from other prokaryotes and eukaryotes.
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Streptomyces griseus Streptomycin Phosphotransferase: Expression of Its Gene in Escherichia coli and Sequence Homology with Other Antibiotic Phosphotransferases and with Eukaryotic Protein Kinases
More LessThe aphD gene of Streptomyces griseus, encoding a streptomycin 6-phosphotransferase (SPH), was sub-cloned in the pBR322-based expression vector pRK9 (which contains the Serratia marcescens trp promoter) with selection for expression of streptomycin resistance in Escherichia coli. Two hybrid plasmids, pCKL631 and pCKL711, were isolated which conferred resistance. Both contained a ~ 2 kbp fragment already suspected to include aphD. The properties of in vitro deletion derivatives of these plasmids were consistent with the presumed location of aphD. In vitro deletion of a sequence including most of the trp promoter largely, but not quite completely, abolished the ability of the plasmid to confer streptomycin resistance, confirming that expression was indeed principally from the trp promoter. A polypeptide of ~ 34·5 kDa was present in minicells containing plasmids that conferred streptomycin resistance, but was absent when the plasmids contained in vitro deletions removing streptomycin resistance. Part of the fragment was sequenced and an open reading frame corresponding to aphD identified. A computer-assisted comparison of the deduced SPH sequence with those of other antibiotic phosphotransferases suggested a common structure A-B-C-D-E, where B and D were conserved between all sequences compared while A, C and E divided between the streptomycin and hygromycin B phosphotransferases on one hand and kanamycin/neomycin ones on the other. A composite sequence database was searched for homologues to consensus matrices constructed from five approximately 12-residue subsequences within blocks B and D. For one subsequence, corresponding to the N-terminal portion of block D, those sequences from the database that yielded the highest homology scores comprised almost entirely either antibiotic phosphotransferases or eukaryotic protein kinases. Possible evolutionary implications of this homology, previously described by other groups, are discussed.
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Cloning of a Thermostable a-Amylase Gene from Thermomonospora curvata and Its Expression in Streptomyces lividans
More LessThe gene from Thermomonospora curvata CCM 3312 coding for thermostable a-amylase (tarn) has been cloned in Streptomyces lividans TK 24 and localized to a 2·6 kb HindIII-BamHI fragment of DNA. The data presented here show that the tarn gene is expressed at a high level in S. lividans and that the protein is efficiently excreted.
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Cloning of the Serratia marcescens recA Gene and Construction of a Serratia marcescens recA Mutant
More LessA recombinant plasmid, pSM2513, containing an 8·5 kb DNA insert was isolated from a genomic library of Serratia marcescens by using interspecific complementation. This plasmid conferred resistance to methyl methanesulphonate and UV irradiation upon recA mutants of Escherichia coli and enhanced recombination proficiency, as measured by Hfr-mediated conjugation, in recA mutants of E. coli. Furthermore, when recA mutants of E. coli harbouring pSM2513 were subjected to UV irradiation, filamentation of the cells was observed. This did not occur upon UV irradiation of the same mutants harbouring the cloning vector alone. These results imply that the S. marcescens recA gene on pSM2513 is functionally similar to the E. coli recA gene in several respects. Restriction enzyme analysis and subcloning studies revealed that the S. marcescens recA gene was located on a 2·7 kb BglII-KpnI fragment of pSM2513, and its gene product of approximately 39 kDa resembled the E. coli RecA protein in molecular mass. Using transformation-mediated marker rescue, a recA mutant of S. marcescens was successfully constructed; its proficiency both in homologous recombination and in DNA repair was abolished compared with its parent.
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Close Relationship of Virulent Bacteriophages of Streptococcus salivarius subsp. thermophilus at Both the Protein and the DNA Level
More LessComparisons of 23 virulent phages of Streptococcus salivarius subsp. thermophilus were made based on morphology, host-range, structural protein analysis. DNA restriction patterns and DNA homology. All the phages had isometric heads (diameters 55–58 nm) and striated tails (lengths 221–275 nm). The genome sizes ranged from 37 to 43 kb. The electrophoretic profiles of the structural proteins were related, with at least two major bands of about 23 and 29 kDa in common for all the phages except three, 𝜙80, 𝜙96 and 𝜙99. These latter phages, which had a particular protein composition (main proteins of 29 and 40 kDa) and high DNA homology between each other, showed only a low DNA homology with the other phages. Extensive DNA homologies were demonstrated by Southern hybridization for all the other phages. All these results suggest a close evolutionary relationship between the phages.
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Transfer and Properties of Some Natural and Suicide Replicons in Pasteurella multocida
More LessWe tested the transfer of several plasmids and transposons from Escherichia coli to Pasteurella multocida by filter mating. Two plasmids, pRKTV5 (pRK2013::Tn7) and pUW964 (pRKTV5::Tn5), were derived from pRK2013 - a narrow-host-range plasmid with the broad-hosfrange IncP conjugation genes. Most P. multocida transconjugants obtained with pRKTV5 had Tn7 insertions in the chromosome but some had insertions of the whole plasmid. By contrast, all the transconjugants obtained with pUW964 had insertions of this plasmid or a deleted variant. pUW964 mediated low-frequency transfer of Tn7 or chromosomal markers between P. multocida strains. Broad-host-range IncP plasmid RP4 (RK2) did not yield selectable transconjugants in P. multocida but two plasmids derived by Tn5 insertion into a kanamycin-sensitive derivative of RP4 did yield transconjugants. pSUP1O11, a narrow-host-range p15A replicon with the RP4 mob region allowing mobilization by the IncP conjugation genes also yielded transconjugants while several other plasmids tested did not transfer markers to P. multocida.
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Restriction Fragment Polymorphism in Mitochondrial DNA of Cryptococcus neoformans
More LessThe restriction patterns of mitochondrial DNA from 20 isolates of the two varieties of Cryptococcus neoformans were compared. The patterns exhibited extensive heterogeneity among the isolates regardless of their serotype or varietal status. Hybridizations with cloned fragments of the conserved cytochrome oxidase gene from Saccharomyces cerevisiae exhibited at least seven patterns among the 20 isolates. There were, however, similarities in the restriction patterns among isolates within the same serotype that were not shared by isolates of other serotypes. Intra-varietal similarities were observed in the restriction patterns among the isolates of C. neoformans var. neoformans which were not present in the restriction patterns among the isolates of C. neoformans var. gattii. Hybridization of some cloned mitochondrial DNA fragments to total DNA digests of various isolates revealed polymorphic as well as variety-specific patterns of homology. These findings agree with the antigenic heterogeneity among the isolates and support he current taxonomic classification of C. neoformans into two varieties.
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Cloning and Sequencing of the gerD Gene of Bacillus subtilis
More LessSummary: A Tn917 insertion in the same region of the chromosome as gerD gave rise to a mutant (ger-97) with a germination phenotype similar to that of two gerD mutants which germinate abnormally in a range of germinants. The insertion and two gerD mutations were cotransformed with ribosomal protein genes rpoB, rpsE and rpsl. DNA cloned from one side of the insertion carried the 16S end of the ribosomal RNA operon rrnl. These data were consistent with the order rpoB-rpsE-rpsI-gerD/ger-97::Tn917-rrnI. Insertion into the wild-type chromosome of a plasmid carrying DNA adjacent to the insertion permitted the recovery of a 1·8 kb fragment of DNA which complemented ger-97::Tn9/7and the gerD mutations. The DNA nucleotide sequence of the region of this fragment at which Tn917 had inserted revealed a 555 bp open reading frame, preceded by a ribosome-binding site and potential σEand σApromoter regions and encoding a predicted polypeptide of 21117 Da. This polypeptide was largely hydrophilic but contained a hydrophobic region at the N-terminus resembling a signal peptide.
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Construction of a Chimeric Series of Bacillus Cyclomaltodextrin Glucanotransferases and Analysis of the Thermal Stabilities and pH Optima of the Enzymes
More LessThe cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from the alkalophilic Bacillus sp. strain no. 171 was cloned in Escherichia coli. The cloned CGTase gene consisted of a single open reading frame which would encode a polypeptide of 713 amino acids, and the first 27 amino acid residues comprised a signal peptide. The nucleotide sequence and the amino acid sequence of this CGTase (CGTase 17-1) gene had strong homology with those of the CGTase (CGTase 38-2) gene previously cloned in our laboratory from the alkalophilic Bacillus sp. strain no. 38-2, although the enzymic properties of the CGTase 17-1 were distinct from those of the CGTase 38-2. To analyse those enzymic properties further, we constructed 12 chimeric CGTases using three restriction nuclease sites and compared the enzymic properties of the chimeric CGTases. The N-terminal part of the enzyme was important for heat stability, and the pH-activity profile was influenced by both the N- and the C-terminal parts. A third segment was less important for enzymic properties.
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- Pathogenicity And Medical Microbiology
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Novel Aerobactin Receptor in Klebsiella pneumoniae
More LessSeveral Klebsiella pneumoniae strains which produced enterochelin but not aerobactin were nevertheless sensitive to cloacin DF13. In contrast, a strain of serotype Kl:O1 which produced both siderophores was cloacin-resistant. Loss by mutation of the O1 but not Kl antigen rendered this strain cloacin-sensitive, indicating that the O1 antigen prevented access of cloacin to the cloacin/aerobactin receptor. Unlike the Kl:O1 strain, the aerobactin-negative strains failed to hybridize in a colony blot assay with an aerobactin receptor gene probe prepared from pColV-K30. However, antisera raised against the 74 kDa pColV-K30 aerobactin receptor cross-reacted with a 76 kDa outer-membrane protein in each K. pneumoniae strain. In addition to the 76 kDa protein, the K1:O1 strain also produced a strongly cross-reacting 74 kDa protein. To determine whether these aerobactin-negative strains could use aerobactin, mutants unable to synthesize siderophores were isolated. Aerobactin promoted the growth of these mutants in iron-deficient media. The evidence presented suggests that some K. pneumoniae strains produce an aerobactin iron-uptake system without apparent production of aerobactin and which is probably based on a 76 kDa receptor, the gene for which does not hybridize with aerobactin receptor gene encoded on pColV-K30.
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Effect of Subculturing on Expression of a Cell-surface Protein Antigen by Streptococcus mutans
More LessTwo freshly isolated strains, Xc and Yc, of Streptococcus mutans serotype c from human dental plaque were subcultured 100 times in Brain Heart Infusion broth. The cell-surface hydrophobicity of strain Xc markedly decreased after subculturing 60 times, but that of strain Yc remained unaltered. Radioimmunoassay showed a close correlation between surface hydrophobicity and the amount of a cell-surface protein antigen (PAc) of M r 190000. One hydrophilic variant (strain Xc100L), one relatively hydrophobic variant (strain Xc100H), and two hydrophobic variants (strains Yc100H1 and Ycl00H2) were isolated from the 100-fold subcultures of hydrophobic strains Xc and Yc, respectively. SDS-PAGE showed that the amount of cell-associated and cell-free PAc of strain Xc100L was smaller than that of strains Xc and Xc100H. Strain Yc100H2 produced larger amounts of cell-associated PAc than strains Yc and Yc100H1. Resting cells of hydrophilic strain Xc100L attached in smaller numbers to saliva-coated hydroxyapatite than did other hydrophobic strains. RNA dot-blot analysis demonstrated a significant decrease in PAc-specific mRNA in strain Xc100L, as compared with strains Xc and Xc100H. Neither rearrangement nor deletion in the structural gene (pac) for PAc of these strains was observed by Southern blot analysis. These findings suggest that a mechanism which regulates the transcription of the pac gene participates in the quantitative variation of PAc after repeated subculturing.
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Antigenic and Structural Analysis of Treponema denticola
More LessPolypeptide and Western immunoblot profiles of subcellular fractions of Treponema denticola ATCC 33520 have been determine by SDS-PAGE of Triton X-100-soluble and -insoluble fractions, a lipopolysaccharide-enriched fraction and purified flagella. Major Triton X-100-soluble polypeptides of 72, 68, 54 and 52 kDa were detected. The 54 kDa polypeptide appeared to be a breakdown product o a larger, heat-modifiable polypeptide. Based on the results o SDS-PAGE analysis and immunoblotting of proteinase K digests of T. denticola, a ‘rough’ lipopolysaccharide appeared to be present. Electron microscopy has been used to monitor the effect of detergent treatment on the morphology o the organism and to examine the detailed structure of the flagella. Treatment with Triton removed the T. denticola outer membrane, resulting in exposure of the flagella. The flagella were shown to have a complex sheath an core structure and polypeptide composition characteristic o that observed for other treponemes. Polypeptides of 38, 35, 32 and 28 kDa were present in purified flagella preparations. Immunoelectron microscopy, iodine-labelling and Western blotting were used to demonstrate the exposure o antigens on the T. denticola surface. Surface iodination located polypeptides of 72, 68 and 54 kDa. Antiserum raised against whole cells of T. denticola recognized these polypeptides and an additional polypeptide of 52 kDa. These data provide a basis for future detaile molecular analysis of the ultrastructure an antigenicity of T. denticola.
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Surface-exposed Epitopes on the Major Outer-membrane Protein of Chlamydia trachomatis Defined with Peptide Antisera
More LessThe surface exposure of computer-predicted, linear B-cell epitopes on the major outer-membrane protein (MOMP) of Chlamydia trachomatis serovar B was assessed using antibodies raised against synthetic peptides in conjunction with immunogold transmission electron microscopy. Several of the chosen peptides elicited antibodies which reacted with both denatured and native MOMP. The majority of the exposed epitopes were found within the variable segments of MOMP. For each of the epitopes identified, the extent of their surface accesssibility varied both among individual organisms and different developmental forms. Evidence for two distinct subspecies-specific epitopes within VS4 is presented.
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Adherence of Multiple Serovars of Chlamydia trachomatis to a Common Receptor on HeLa and McCoy Cells Is Mediated by Thermolabile Protein(s)
More LessSeveral aspects of the adherence of purified elementary bodies (EB) of Chlamydia trachomatis to HeLa and to McCoy cells were examined using different techniques, including an ELISA. Serovar-specific, biotinylated monoclonal antibodies were used to detect cell-bound chlamy-diae. In addition, purified chlamydiae were biotinylated and their adherence properties were studied. The assays were done at 4°C to exclude the energy-dependent internalization of the cell-bound EB and host-cell membrane recycling that occur at 37°C. Saturation kinetics were routinely observed at 4°C, and the rate of adherence remained linear for approximately 60 min. Lineweaver-Burk analysis of the kinetics data showed that adherence of any one serovar was competitively inhibited by other serovars of C. trachomatis. This competition for the same receptor on the two alternative hosts, HeLa and McCoy, was also seen when the adherence assays were done at 37°C in the presence of sodium azide, an energy poison that inhibits endocytosis of cell-bound chlamydiae. Chlamydiae exposed to 56°C for 5 min, or treated with low doses of trypsin, failed to exhibit competitive inhibition, having suffered considerable loss of the ability to adhere to host-cells. These data suggest that heat- and trypsin-labile chlamydial moieties participate in the adherence reaction, and that oculo-genital serovars of C. trachomatis, including that of lymphogranuloma venereum, attach to the same receptor on the host-cell membrane.
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Identification of Epitopes Recognized by Monoclonal Antibodies SM1 and SM2 Which React with All Pili of Neisseria gonorrhoeae but Which Differentiate between Two Structural Classes of Pili Expressed by Neisseria meningitidis and the Distribution of Their Encoding Sequences in the Genomes of Neisseria spp.
More LessThe pili expressed by all isolates of Neisseria gonorrhoeae react with two monoclonal antibodies, SM1 and SM2. In contrast, although many isolates of Neisseria meningitidis also express pili (class I) which react with antibodies SM1 and SM2, a proportion express pili (class II) which fail to react. In order to define the epitopes recognized by these antibodies, a series of overlapping peptides corresponding to the amino acid sequence of conserved regions of gonococcal pili have been synthesized. The minimum epitope recognized by antibody SM1 was found to comprise a linear peptide EYYLN, corresponding to residues 49–53 of mature pilin. In contrast, antibody SM2 reacted with a number of peptides from around the cysteine residue (Cys 1) at position 120, suggesting that an extended region may contribute to a conformational epitope recognized by this antibody in the native protein. The identification of the two epitopes defines structural differences between the classes of pili expessed by meningococci. In order to determine the distribution of pilin gene sequences in Neisseria we used as hybridization probes an oligonucleotide (PS1) with the sequence 5′-GAGTATTACCTGAATCA-3' which spans the coding region for the SM1 epitope, and a fragment of the 3′ end of the gonococcal pilE gene which contains conserved sequences flanking the two Cys codons and encodes the SM2 epitope. All strains of N. gonorrhoeae and N. meningitidis tested, regardless of piliation phenotype, harboured DNA sequences homologous to those encoding the carboxy-terminus of meningococcal class I pilin. Furthermore, all gonococci and all meningocococci producing class I pili hybridized with oligonucleotide probe PS1. Non-reverting non-piliated derivatives of previously class I pilus-producing strains showed reduced hybridization signals with this probe, but nevertheless retained sequences homologous to the coding sequence for the SM1 epitope. However, meningococci producing class II pili could be divided into two groups on the basis of their reaction with the PS1 probe: half the strains tested failed to react, which is consistent with our previous analysis of silent class I pilin sequences; the remainder reacted (relatively weakly) with the probe, suggesting that the silent pil sequences in these strains extend further towards the 5′ end of the pilin gene than in strains studied previously. Some strains of Neisseria lactamica reacted weakly with both types of probe but failed to produce SM1-reactive pili. In contrast,
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Plasmid-mediated Chloramphenicol Resistance in Staphylococcus hyicus
More LessA small plasmid of 3·95 kb, encoding resistance to chloramphenicol (Cm) was detected in three of 33 Staphylococcus hyicus strains. The plasmid in each of the three strains was indistinguishable by Southern-blot hybridization and restriction enzyme analysis. It was shown by curing and by transformation to specify resistance to Cm. A preliminary restriction map of the plasmid, designated pSC2, is presented. Chloramphenicol acetyltransferase was demonstrated by enzyme assay and by SDS-PAGE of cell-free lysates of pSC2 transformants.
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Biological Attributes of Colony-type Variants of Candida albicans
More LessTwenty ‘commensal’ oral or ‘pathogenic’ vaginal isolates of Candida albicans were examined for colony morphology on malt/yeast-extract and serum-based agar media. Diverse and variable colony morphology was seen on serum agar. In 17 strains, selective subculture of morphologically atypical colonies produced progeny which had reverted to the morphology of the majority of parental colonies. However, in one strain, a highly stable colony variant was isolated which did not revert on subculture. In two further strains, variants were isolated which could be maintained with at least 99 % homogeneous colony type by selective colony subculture, but reversion to the parental type or switching to other morphologies occurred at rates of 10−2 to 10−4: a rapid switching phenomenon. The relative proportions of mycelial or yeast forms were the main determinants of colony morphology. The variants were biotyped using a selection of biochemical tests. The stable variant differed from its parent in several characters, including rate of production of a proteinase enzyme. The pathogenicity of variants was compared in mice, and both stable and switching variants differed in virulence from their parental strains. Colony-type variation on suitable media is thus a powerful tool in the isolation of mutants or variants of C. albicans which differ from ‘isogenic’ parents in significant biological properties. Such variants may aid identification and characterization at the molecular level of determinants of, for example, pathogenicity and morphogenesis.
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- Physiology And Growth
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Inactivation of Coxiella burnetii by Gamma Irradiation
More LessThe gamma radiation inactivation kinetics for Coxiella burnetii at − 79°C were exponential. The radiation dose needed to reduce the number of infective C. burnetii by 90% varied from 0·64 to 1·2 kGy depending on the phase of the micro-organism, purity of the culture and composition of suspending menstruum. The viability of preparations containing 1011C. burnetii ml−1was completely abolished by 10 kGy without diminishing antigenicity or ability to elicit a protective immune response in vaccinated mice. Immunocytochemical examinations using monoclonal antibodies and electron microscopy demonstrated that radiation doses of 20 kGy did not alter cell-wall morphology or cell-surface antigenic epitopes.
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Characterization of a Novel Yeast Synthesizing Melanin-like Pigment
More LessA laboratory yeast isolate identified as Exophiala jeanselmei synthesized melanin and showed polyphenol oxidase activity with dihydroxyphenylalanine (DOPA) and pyrogallol. Electrophoresis on native and denaturing gels, and activity staining with DOPA revealed two immunologically distinguishable bands of Mr about 100000 and 120000, which could be separated by (NH4)2SO4 fractionation. The enzyme isolated from this yeast gave optimal activity at higher pH (8·5–10) and temperature (37–40 °C) than other known melanin-synthesizing activities.
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Growth and Lipid Composition of Some Dematiaceous Hyphomycete Fungi Grown at Different Salinities
More LessThe growth of the dematiaceous hyphomycetes (darkly pigmented microfungi) Alternaria phragmospora, Alternaria chlamydospora and Ulocladium chartarum was altered by increasing concentrations of NaCl in the growth medium. U. chartarum demonstrated the greatest sensitivity to salinity changes: chlamydospora-like structures were common at NaCl concentrations > 5 %. Lipid analyses of these fungi are presented. Steryl esters and fatty acids were the main lipid classes in the apolar fractions. Phosphatidylethanolamine, phosphatidylgly-cerol and phosphatidylcholine were the main polar compounds. Most phospholipids decreased at higher salinity. The major fatty acid was oleic acid (18:1) which decreased at 10 % NaCl. A similar pattern was observed for palmitic acid (16:0). Myristic acid (14:0) and myristoleic acids (14:2, 14:1) were detected in all isolates cultured at 10 % NaCl.
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Comparison of the Uptake Systems for the Entry of Various BtuB Group Colicins into Escherichia coli
More LessColicins A, E1, E2 and E3 belong to the BtuB group of colicins. The NH2-terminal region of colicin A is required for translocation, and defects in this region cannot be overcome by osmotic shock of sensitive cells. In addition to BtuB, colicin A requires OmpF for efficient uptake by sensitive cells. The roles of BtuB and OmpF in translocation and binding to the receptor of the colicins A, E1, E2 and E3 were compared. The results suggest that for colicin A OmpF is used both as a receptor and for translocation across the outer membrane. In contrast, for colicin E1, OmpF is used neither as a receptor nor for translocation. For colicins E2 and E3, the situation is intermediate: only BtuB is used as a receptor but both BtuB and OmpF are involved in the translocation step.
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Production of the Fimbrial Adhesin 987P by Enterotoxigenic Escherichia coli during Growth under Controlled Conditions in a Chemostat
More LessThe effects of growth conditions on the production of 987P fimbriae by the enterotoxigenic Escherichia coli strain 1592 were examined in steady state chemostat experiments at different specific growth rates. The amount of fimbriae produced by fimbriate cells (P+) was dependent on the specific growth rate (μ). Under aerobic growth conditions fimbriae production increased with higher μ values till μ = 0·40 h−1and decreased again at μ values close to μ max (0·48 h−1). Under anaerobic growth conditions the maximal production was comparable to that under aerobic growth conditions, and was also maximal close to μ max (0·16 h−1). Phase variation, measured as the percentage of fimbriate cells in a particular population, was independent of μ. The composition of the growth medium influenced both phase variation and overall production of fimbriae. A shift from minimal to a complex medium induced a rapid reduction in the amount of fimbriae per P+cell and a slower reduction in the percentage of P+cells. A shift from complex to minimal medium resulted in an increase in the percentage of P+cells and a constant amount of fimbriae per P+cell. The frequency of the phase switch was calculated for different growth conditions. The frequency of the P+ → P−switch between two steady states was 2·7 × 10−2. In batch culture the frequency of the P− → P+switch was minimally 2·9 x 10−2. The results indicate that phase variation and the production of 987P fimbriae by fimbriate cells are under independent physiological control.
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Isolation and Characterization of a Mutant of Trichoderma reesei Showing Reduced Levels of Extracellular β-Glucosidase
Saroj Mishra, S. Rao and J. K. DebA mutant (D6-13) of Trichoderma reesei that produced low levels of extracellular β-glucosidase was physiologically and biochemically characterized. The mutant was unable to grow and produced very little cellulase activity on Solka-Floc, Avicel, micro-crystalline cellulose and acid-swollen cellulose. Whereas growth was nearly normal on cellobiose, lactose and glucose, cellulase production was slightly decreased on cellobiose and lactose. The mutant produced nearly normal levels of FPA (filter-paper activity) and endoglucanase on sophorose. A distinct lag and a lowered specific sugar consumption rate was observed for the mutant on 0·3% cellobiose; this appeared to be due to lowered wall-bound β-glucosidase activity. The multiplicity and role of different β-glucosidases in T. reesei is discussed.
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Analysis of Periplasmic Enzymes in Intact Cultured Bacteria and Bacteroids of Bradyrhizobium japonicum and Rhizobium leguminosarum biovar phaseoli
More LessAnalysis of periplasmic enzymes of Bradyrhizobium japonicum or Rhizobium leguminosarum biovar phaseoli was hampered by the fact that only small amounts of marker enzyme activities were released from cells by osmotic shock or by lysozyme/EDTA treatment. However, up to 95 % of total activity of certain periplasmic marker enzymes could be measured in intact cells by including 003% Triton X-100 in enzyme assays. Less than 5% of the cytoplasmic marker enzyme -hydroxybutyrate dehydrogenase could be measured under these conditions, indicating that treatment with dilute detergent does not significantly perturb the cytoplasmic membrane. Various lines of evidence indicate that the dilute detergent treatment does not significantly alter wall structure but, instead, permits the assay of periplasmic enzymes by facilitating diffusion of substrates and products to and from the periplasmic space. For pyrophosphatase and phosphodiesterase, the proportion of total enzyme activity which could be measured in intact cells was higher in bacteroids than in bacteria at all Triton concentrations, suggesting that the outer membrane of bacteroids may be more permeable than the outer membrane of cultured bacteria. The effect of detergent on the hydrolysis of four disaccharides sucrose, α,α-trehalose, maltose and lactose was studied using intact cultured R. leguminosarum biovar phaseoli. The response of lactose hydrolysis to the addition of detergent was four-fold greater than the response of the other disaccharidase activities, indicating that a rhizobial lactase may be present in the periplasmic space.
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Effect of Bicarbonate on the Growth of Actinobacillus actinomycetemcomitans in Anaerobic Fructose-limited Chemostat Culture
More LessThe effect of bicarbonate on the growth and product formation by a periodontopathic bacterium, Actinobacillus actinomycetemcomitans, was examined in an anaerobic chemostat culture with fructose as the limiting nutrient. The chemostat cultures were run at dilution rates between 0·04 and 0·25 h−1and the maximum growth yield (Y fructose max) was estimated to be 40·3 and 61·7 g dry wt (mol fructose)−1in the absence and presence of bicarbonate, respectively. The major fermentation products in the absence of bicarbonate were formate, acetate, ethanol and succinate, with small amounts of lactate. The addition of bicarbonate to the medium resulted in a marked decrease in ethanol production and in a significant increase in succinate production. Washed cells possessed activity for the cleavage of formate to CO2 and H2, which seemed to play a role in supplying CO2 for the synthesis of succinate in the absence of bicarbonate. The study of enzyme activities in cell-free extracts suggested that fructose was fermented by the Embden-Meyerhof-Parnas pathway. The values of Y ATP maxand the efficiency of ATP generation (ATP-Eff) during fructose catabolism were estimated and higher values were obtained for the culture in the presence of bicarbonate: 20·2 g dry wt (mol ATP)−1and 30 mol ATP (mol fructose)−1, respectively, versus Y ATP max = 15·1 and ATP-Eff = 2·7 in the absence of bicarbonate.
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Accumulation of Polyhydroxy Alcohols by Hansenula anomala in Response to Water Stress
More LessThe response of the yeast Hansenula anomala to a reduction in water activity (a w) from 0·998 to 0·925 (adjusted with glucose or NaCl) was monitored. Natural abundance 13C NMR spectroscopy and HPLC analysis revealed that the type of carbon source determined which polyols were present intracellularly at 0·998 a w. At 0·95 a w (NaCl), glycerol was accumulated in all instances irrespective of the type of carbon source indicating the primary role of glycerol in osmoregulation. The carbon source had a bearing only on which other polyol(s) were accumulated. During growth on glucose at 0·95 a w (NaCl or glucose), glycerol was accumulated intracellularly in the exponential growth phase with a concentration ratio (intra-/extracellular) as high as 10000-fold whereas during the stationary phase arabitol accumulation occurred to a lower concentration ratio while the glycerol concentration decreased. The specific growth rate and cell volume decreased with increasing NaCl or glucose concentrations. This indicated that the yeast had no specific requirement for these compounds for optimum growth but tolerated high concentrations. Reducing the a w to 0·95 resulted in increasing intracellular concentrations of glycerol and arabitol whereas below 0·95 a W (NaCl or glucose), the intracellular polyol concentration decreased while the polyol concentration ratios across the cell membrane increased. During the early exponential growth phase at 0·95 a w, glycerol was accumulated in sufficiently high concentrations to achieve an osmotic balance across the membrane whereas in the stationary phase the arabitol and glycerol concentration was insufficient to maintain the osmotic balance.
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- Systematics
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Bilophila wadsworthia, gen. nov. and sp. nov., a Unique Gram-negative Anaerobic Rod Recovered from Appendicitis Specimens and Human Faeces
Strongly catalase-positive Gram-negative anaerobic rods were isolated from approximately half of all intra-abdominal specimens received from patients with gangrenous and perforated appendicitis, and subsequently also from normal faecal specimens. The organism was originally detected on Bacteroides-bile-aesculin (BBE) agar, and grew slowly on non-selective anaerobic media containing blood. It was stimulated by bile and differed from other known genera by being urease- and catalase-positive, and by reducing nitrate. It did not reduce sulphate. Other anaerobic Gram-negative rods showed no homology by DNA dot-blot hybridization. The thermal melting profile of chromosomal DNA showed 39–40 mol% G + C. The whole-cell fatty acid methyl ester profile included cyclic and branched long-chain acids, and differed from those of all other anaerobes that have been tested.#x03B2;-Lactamase was not detected. The name Bilophila wadsworthia gen. nov., sp. nov. is proposed for this organism.
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