- Volume 143, Issue 10, 1997
Volume 143, Issue 10, 1997
- Review Article
-
- Antigens And Immunity
-
-
-
Peptides 14VIDLL18 and 96FEAAAL101 defined as epitopes of antibodies raised against amino acid sequences of enterotoxigenic Escherichia coli colonization factor antigen I fused to Salmonella flagellin
More LessAntibodies raised against four hybrid Salmonella flagellins carrying amino acid sequences derived from the fimbrial subunit of the colonization factor I antigen (CFA/I) of enterotoxigenic Escherichia coli (ETEC), i.e. hybrid flagellins Fla I (aa 1-15), Fla II (aa 11-25), Fla III (aa 32-45) and Fla IV (aa 88-102), were not able to inhibit the in vitro binding of CFA/I-expressing ETEC bacteria to enterocyte-like Caco-2 cells. However, one of the hybrid flagellins (Fla II) was recognized by a previously described anti-CFA/I subunit mAb (S-CFA/I 17:8) which was able to block adhesion of CFA/l-expressing bacteria to Caco-2 cells and to bind to the amino acid sequence 15IDLLQ19 of the CFA/I fimbrial subunit. Pepscan analysis of antibodies raised against the hybrid flagellins Fla II and Fla IV showed that they were specific for the sequences 14VIDLL18 and 96FEAAAL101, respectively, of the CFA/I fimbrial subunit. Thus, the discrepancy in the abilities of the anti-Fla II serum and the mAb S-CFA/I 17:8 to block binding might be ascribed to their slightly different fine specificity for epitopes.
-
-
-
-
The immunoreactive 116 kDa surface protein of Mycoplasma pneumoniae is encoded in an operon
More LessSera from 10 patients infected with Mycoplasma pneumoniae were used in Western blot analysis of Triton-X-114-soluble protein preparations of M. pneumoniae. All 10 sera were reactive with a protein antigen of 116 kDa. Sera from another 17 patients were used in Western blot analysis of whole-cell M. pneumoniae proteins; 15 of these sera were reactive with the 116 kDa protein. Trypsin digestion of whole M. pneumoniae cells demonstrated the surface location of this protein. Sequencing of DNA which contained the gene for this protein identified an ORF of 3093 bp encoding a protein with a predicted molecular mass of 116013 Da. The ORF for the 116 kDa protein had 99·8% nucleotide identity with the M. pneumoniae gene G07_orf1030 and 61% nucleotide identity with the Mycoplasma genitalium ORF MG075 of unassigned function. An ORF which was identified 5' to the 116 kDa protein ORF coded for a 16 kDa protein and had 99·8% nucleotide identity with the M. pneumoniae gene G07_orf135 and 58·4% nucleotide identity with the ORF MG074 of M. genitalium. Analysis of mRNA detected a 3·7 kb transcript with a single initiation site 5' to the ORF encoding the 16 kDa protein. The coding sequences for both the 16 kDa protein and the 116 kDa protein were present in this transcript, indicating that they were part of an operon and suggesting a possible functional relationship.
-
- Biochemistry
-
-
-
Fungicides and sterol-deficient mutants of Ustilago maydis: plasma membrane physico-chemical characteristics do not explain growth inhibition
More LessPlasma membrane vesicles from erg11 and erg2 sterol-deficient mutants and from wild-type Ustilago maydis sporidia treated with and without inhibitors of sterol 14α-demethylase or sterol ∆8–∆7 isomerase (triadimenol and fenpropimorph fungicides, respectively) were purified by aqueous two-phase partitioning. Changes in plasma membrane lipid composition were mostly restricted to sterols and complex lipid-bound fatty acids (CLB fatty acids). There was a greater accumulation of abnormal sterols (14α-methyl-or ∆ 8-unsaturated sterols) in plasma membranes from sterol-deficient mutants than from those treated with their fungicide counterparts. However, greater growth inhibition was observed on fungicide-treated wild-type than on mutants. Changes in CLB fatty acids were restricted to alterations in the relative proportion of linoleic acid (18:2) with respect to oleic acid (18:1). The 18:2 to 18:1 ratio found in CLB fatty acids in plasma membranes could be correlated to rates of sporidial growth but not to accumulation of a particular abnormal sterol or to the extent of sterol replacement. Plasma membrane permeability to protons was increased moderately in the mutants only. No changes were observed in plasma membrane fluidity. Plasma membrane H+-ATPase activity was increased up to twofold in those cases with lower growth rate. It was concluded that fungicide-induced growth inhibition in U. maydis was not due to accumulation of abnormal sterols in plasma membranes but probably due to intracellular ATP depletion by the H+-ATPase and that changes in 18:2 to 18:1 ratio in CLB fatty acids were not directly dependent on the plasma membrane physical state or lipid composition but were possibly part of a stress adaptation mechanism.
-
-
- Biotechnology
-
-
-
Recombinant SINAP-25 is an effective substrate for Clostridium botulinum type A toxin endopeptidase activity in vitro
More LessBacterial neurotoxins are now being used routinely for the treatment of neuromuscular conditions. Alternative assays to replace or to complement in vivo bioassay methods for assessment of the safety and potency of these botulinum neurotoxin-based therapeutic products are urgently needed. Advances made in understanding the mode of action of clostridial neurotoxins have provided the basis for the development of alternative mechanism-based assay methods. Thus, the identification of SNAP-25 (synaptosomal-associated protein of molecular mass 25 kDa) as the intracellular protein target which is selectively cleaved during poisoning by botulinum neurotoxin type A (BoNT/A) has enabled the development of a functional in vitro assay for this toxin. Using recombinant DNA methods, a segment of SNAP-25 (aa residues 134-206) spanning the toxin cleavage site was prepared as a fusion protein to the maltose-binding protein in Escherichia coli. The fusion protein was purified by affinity chromatography and the fragment isolated after cleavage with Factor Xa. Targeted antibodies specific for the N and C termini of SNAP-25, as well as the toxin cleavage site, were prepared and used in an immunoassay to demonstrate BoNT/A endopeptidase activity towards recombinant SNAP-25 substrates. The reaction required low concentrations of reducing agents which were inhibitory at higher concentrations as were metal chelators and some inhibitors of metallopeptidases. The endopeptidase assay has proved to be more sensitive than the mouse bioassay for detection of toxin in therapeutic preparations. A good correlation with results obtained in the in vivo bioassay (r = 0·95, n = 23) was demonstrated. The endopeptidase assay described here may provide a suitable replacement assay for the estimation of the potency of type A toxin in therapeutic preparations.
-
-
- Genetics And Molecular Biology
-
-
-
Disruption of the Pseudomonas aeruginosa dipZ gene, encoding a putative protein-disulfide reductase, leads to partial pleiotropic deficiency in c-type cytochrome biogenesis
More LessThe Pseudomonas aeruginosa dipZ gene has been cloned and sequenced. Whereas disruption of Escherichia coli dipZ (dsbD), the hydrophilic C-terminal domain of which has been deduced to be periplasmic and to function as a protein-disuifide reductase, leads to the absence of c-type cytochromes, disruption of P. aeruginosa dipZ attenuated, but did not abolish, holo-c-type cytochrome biosynthesis. Comparison of the P. aeruginosa DipZ sequence with three other DipZ sequences indicated that there are not only two conserved cysteine residues in the C-terminal hydrophilic domain, but also two more in the central highly hydrophobic domain. The latter would be located toward the centre of two of the eight membrane-spanning α-helices predicted to compose the hydrophobic central domain of DipZ. Both these cysteine residues, plus other transmembrane helix residues, notably prolines and glycines, are also conserved in a group of membrane proteins, related to Bacillus subtilis CcdA, which lack the N- and C-terminal hydrophilic domains of the DipZ proteins. It is proposed that DipZ of P. aeruginosa and other organisms transfers reducing power from the cytoplasm to the periplasm through reduction and reoxidation of an intramembrane disulfide bond, or other mechanism involving these cysteine residues, and that this function can also be performed by B. subtilis CcdA and other CcdA-like proteins. The failure of dipZ disruption to abolish c-type cytochrome synthesis in P. aeruginosa suggests that, in contrast to the situation in E. coli, the absence of DipZ can be compensated for by one or more other proteins, for example a CcdA-like protein acting in tandem with one or more thioredoxin-like proteins.
-
-
-
-
A geographically widespread plasmid from Thiobacillus ferrooxidans has genes for ferredoxin-, FNR-, prismane- and NADH-oxidoreductase-like proteins which are also located on the chromosome
More LessDuring a search for genes encoding electron transport proteins from a Thiobacillus ferrooxidans ATCC 33020 gene bank, a 19.8 kb plasmid, pTF5, which conferred increased sensitivity to the antimicrobial agent metronidazole upon an Escherichia coli mutant, was isolated and cloned in E. coli. The plasmid had an identical restriction enzyme map to a plasmid which has been found in T. ferrooxidans strains isolated from many different parts of the world. The plasmid was present at between two and four copies per genome and contained a region of approximately 5.6 kb which was also found on the chromosome. This region was sequenced and found to have four complete ORFs, which when translated had high percentage amino acid similarity to [3Fe-4S,4Fe-4S] ferredoxins, proteins of the FNR regulator family, prismane-like proteins and the NADH oxidoreductase subunit of a methane monooxygenase. In vitro protein analysis using an E. coli-derived transcription-translation system indicated that three of the four products (FdxA, PsmA and RedA) were expressed in the heterologous system. Ferredoxins, prismane-like proteins and NADH oxidoreductases are redox-active proteins and it is likely that the proteins on pTF5 represent an electron transport system of as yet unknown function. Surprisingly, although genes for redox-active proteins have been isolated from other bacteria by screening gene banks for increased sensitivity to metronidazole, the region of pTF5 containing the genes for these proteins was not responsible for the increase in metronidazole sensitivity conferred by the plasmid. The region of pTF5 which did confer increased metronidazole sensitivity to an E. coli metronidazole-resistant mutant was a 319 bp region of DNA close to the origin of plasmid replication. This region contained no ORFs and was identical to that previously reported for the replicon of a 9.8 kb T. ferrooxidans plasmid, pTFI91.
-
-
-
Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors
The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cmr derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non-selective conditions.
-
-
-
Glyceraldehyde-3-phosphate dehydrogenase expression in Trichoderma harzianum is repressed during conidiation and mycoparasitism
More LessA glyceraldehyde-3-phosphate dehydrogenase (gpd) cDNA was isolated from the filamentous fungus Trichoderma harzianum in the course of a search for light-regulated genes in this organism. There is apparently only one copy of gpd in the T. harzianum genome, and its sequence is most similar to that of other filamentous ascomycetes. Trichoderma grows in the soil as a saprophyte or mycoparasite. A brief pulse of blue light, or nutrient depletion, induces sporulation, which is accompanied by altered patterns of abundance of specific polypeptides. Mycoparasitic development is also accompanied by changes in gene expression. The abundance of gpd mRNA decreased strongly during sporulation, and was lowest in samples consisting of mature conidiophores and conidia. When T. harzianum was grown in the presence of cell walls of the phytopathogen Rhizoctonia solani, the gpd mRNA level was much lower than in similar cultures grown on glucose. The repression of gpd, which is usually considered a constitutively expressed gene, may be part of the switch to sporulation or to the simulated mycoparasitic state. The implications of these findings for the use of gpd promoters to confer high constitutive expression are discussed.
-
-
-
Binding of colicins A and E1 to purified ToIA domains
Colicins are divided into two groups according to the proteins required for their import into sensitive bacteria. The Tol and TonB pathways are involved in import of group A and group B colicins respectively. Because previous analyses have shown that colicin E1 and colicin A (two group A colicins) interact in vitro with the C-terminal domain of TolA (TolAIII) while colicin B (group B colicin) does not, attention was focused on these interactions with purified proteins. TolA has been described as a three-domain protein with an N-terminal inner-membrane anchor and a long periplasmic region formed by two domains (TolAII and TolAIII). TolAIII, TolAII and TolAII-III soluble domains with an N-terminal hexa-histidine extension were purified. The interactions of colicins with the purified TolA domains were analysed by overlay Western blotting, which indicated that both N-terminal domains of colicins A and E1 interacted with TolAIII, while a gel shift procedure detected no interaction with colicin E1. The binding kinetic values of the N-terminal domains of colicins A and E1 to TolAIII were estimated by surface plasmon resonance and were shown to be similar.
-
-
-
Genetic identification of chemotactic transducers for amino acids in Pseudomonas aeruginosa
More LessTwo chemotactic transducer genes (termed pctB and pctC and an open reading frame (orf1) were found in the pctA-flanking region which was previously identified as a chemotactic transducer gene in Pseudomonas aeruginosa. The pctB and pctC genes encode predicted polypeptides of 629 and 632 amino acids, respectively. Overall, PctB and PctC had 81 and 75% amino acid identities with PctA, respectively. A null mutant strain PCT2, which contained a deletion in the entire pcfC, orf1, pctA and pctB genes, did not show chemotaxis towards all 20 commonly occurring L-amino acids. This mutant strain also failed to respond to amino acid catabolites (cadaverine, 4-aminobutyrate and putrescine) that are strong attractants for the wild-type strain PAO1. To study the role of each gene product in L-amino acid taxis, plasmids harbouring the pctC, orf1, pctA, or pctB genes were constructed and introduced into strain PCT2 by transformation. The orf1 gene did not complement the defect in chemotaxis of strain PCT2. The pctA gene restored the ability of strain PCT2 to respond to 18 L-amino acids, suggesting that PctA plays a major role in detecting L-amino acids in P. aeruginosa. The pctB and pctC genes complemented the defect in chemotaxis to only seven (Ala, Arg, Glu, Lys, Met, Tyr, Gin) and two (His, Pro) L-amino acids, respectively.
-
-
-
Functional and genetic characterization of mcpC, which encodes a third methyl-accepting chemotaxis protein in Bacillus subtilis
More LessA 3135 bp DNA segment downstream of the spl gene on the Bacillus subtilis chromosome was cloned and its nucleotide sequence determined. An open reading frame capable of encoding a putative protein of 654 amino acids with a calculated molecular mass of 72.1 kDa was identified. The deduced amino acid sequence was similar to the McpA and McpB proteins of B. subtilis. McpA and McpB encode different methyl-accepting chemotaxis proteins (MCPs). A mutant strain containing an antibiotic resistance DNA cassette inserted into the region containing the MCP-like reading frame suffered a complete loss of taxis to the amino acids cysteine, proline, threonine, glycine, serine, lysine, valine and arginine. The open reading frame was designated mcpC. The wild-type and an mcpC mutant strain were analysed for their content of methylated proteins and it was found that mcpC encodes a methylated membrane protein that has previously been designated H3. These results show that mcpC encodes a third MCP in B. subtilis. The transcription start site upstream of the mcpC gene was determined by primer extension analysis and it was found to be preceded by a potential promoter sequence that is recognized by the β D form of RNA polymerase. The level of β-gaiactosidase expressed from a transcriptional mcpC-lacZ fusion was increased threefold when cells entered the stationary phase. No β-gaiactosidase could be detected in a sigD genetic background.
-
-
-
High-osmolarity signalling in Saccharomyces cerevisiae is modulated in a carbon-source-dependent fashion
More LessHigh-osmolarity-induced expression of the small heat-shock gene HSP12 is regulated by the HOG (high-osmolarity glycerol) pathway and PKA (protein kinase A). To analyse the regulatory input of both signal transduction pathways, high-salt-induced HSP12 expression in different genetic backgrounds on glucose-, ethanol- and glycerol-based culture media was examined. Upon exposure to high-osmolarity stress, the kinetics of induction of HSP12 in cells growing on the non-fermentable carbon sources are strikingly different from those on glucose. Derepression of HSP12 gene expression under non-stress conditions was observed in cells growing on non-fermentable carbon sources. High-salt challenge resulted in a lower induction of the HSP12 mRNA levels in ethanol-grown cells as compared to glucose-grown cells, whereas in glycerol-grown cells hardly any high-salt induction of HSP12 mRNA levels could be detected. Analysis of signalling through the HOG pathway suggested that glycerol may influence the activity of this signalling route, possibly via negative feedback. Furthermore, the cellular level of PKA activity was found to have a great impact on stress-responsive gene transcription. On the basis of the data obtained it was concluded that modulation of PKA activity plays a major role in the stress response. A glucose-dependent, PKA-regulated cellular component is postulated to affect high-osmolarity-induced HSP12 expression.
-
-
-
Sequencing and mutagenesis of genes from the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea that are involved in L-mycarose and D-desosamine production
The nucleotide sequence on both sides of the eryA polyketide synthase gene of the erythromycin-producing bacterium Saccharopolyspora erythraea reve the presence of ten genes that are involved in L-mycarose (eryB) and D-desosamine (eryC) biosynthesis or attachment. Mutant strains carrying targeted lesions in eight of these genes indicate that three (eryBIV, eryBV an eryBVI) act in L-mycairose biosynthesis or attachment, while the other five (eryCII, eryCIII, eryCIV, eryCV and eryCVI) are devoted to D-desosamine biosynthesis or attachment. The remaining two genes (eryBII and eryBVII) appear to function in L-mycarose biosynthesis based on computer analysis an earlier genetic data. Three of these genes, eryBII, eryCIII and eryCII, lie between the eryAIII and eryG genes on one side of the polyketide synthase genes, while the remaining seven, eryBIV, eryBV, eryCVI, eryBVI, eryCIV, eryC and eryBVII lie upstream of the eryAI gene on the other side of the gene cluster. The deduced products of these genes show similarities to: aldohexos 4-ketoreductases (eryBIV), aldoketo reductases (eryBII), aldohexose 5-epimerases (eryBVII), the dnmT gene of the daunomycin biosynthetic pathwa of Streptomyces peucetius (eryBVI), glycosyltransferases (eryBV and eryCIII), the AscC 3,4-dehydratase from the ascarylose biosynthetic pathway of Yersin pseudotuberculosis (eryCIV), and mammalian N-methyltransferases (eryCVI). The eryCII gene resembles a cytochrome P450, but lacks the conserved cysteir residue responsible for coordination of the haem iron, while the eryCV gene displays no meaningful similarity to other known sequences. From the predicted function of these and other known eryB and eryC genes, pathways for the biosynthesis of L-mycarose and D-desosamine have been deduced.
-
-
-
Dipeptidyl aminopeptidase processing and biosynthesis of alkaline extracellular protease from Yarrowia lipolytica
More LessAlkaline extracellular protease (AEP) from Yarrowia lipolytica is synthesized as a precursor with a 157 aa prepro-region. Signal peptide cleavage was shown to occur after Ala15 by N-terminal amino acid radiosequencing of the largest intracellular AEP precursor. AEP proteolytic activity was not required for AEP processing. After a change of the putative active site Ser to Ala, inactive AEP with the same mobility on SDS-PAGE as wild-type mature AEP was secreted. The role of dipeptidyl aminopeptidase (DPAPase) activity in AEP processing was also investigated. Mutations early in the -X-Ala- and -X-Pro- dipeptide stretch (Pro17 to Met which should prevent DPAPase processing and Ala19 to Val which should allow removal of only the first dipeptide) did not prevent synthesis of active mature AEP nor did use of the DPAPase inhibitor Pro-boroPro. Deletion of the entire dipeptide stretch (Ala16 to Pro33) resulted in intracellular accumulation of an AEP precursor, which surprisingly was not glycosylated, and little or no secretion of AEP-related polypeptides. Expression of AEP in wild-type and dpp1 dap2 Saccharomyces cerevisiae strains (lacking both the Golgi and vacuolar DPAPases) resulted in secretion of only mature AEP and no AEP precursors. Transit times and levels of AEP secretion were similar for both strains. These results indicate that the KEX2-like cleavage after Lys156-Arg157, which yields mature active AEP can occur in the absence of DPAPase processing and that DPAPase processing is not necessary for secretion of mature active AEP.
-
-
-
Identification of new regulatory genes controlling synthesis of folate-dependent enzymes in Aspergillus nidulans
More LessPrototrophic revertants of a metH2 strain of Aspergillus nidulans which is impaired in the regulation of synthesis of folate-dependent enzymes were isolated and six of them analysed. In three of the isolates reversion was the result of an intragenic suppressor mutation in the metH locus. In the remaining strains suppressor mutations occurred in independent genes. These genes, designated folA, folB and folC, are linked and located in chromosome VI. Mutations in these genes render synthesis of some folate enzymes, particularly folylpolyglutamate synthetase, insensitive to methionine-mediated repression.
-
-
-
Molecular analysis of a Clostridium butyricum NCIMB 7423 gene encoding 4-α-glucanotransferase and characterization of the recombinant enzyme produced in Escherichia coli
More LessAn Escherichia coli clone was detected in a Clostridium butyricum NCIMB 7423 plasmid library capable of degrading soluble amylose. Deletion subcloning of its recombinant plasmid indicated that the gene(s) responsible for amylose degradation was localized on a 1·8 kb NspHI-Scal fragment. This region was sequenced in its entirety and shown to encompass a large ORF capable of encoding a protein with a calculated molecular mass of 57184 Da. Although the deduced amino acid sequence showed only weak similarity with known amylases, significant sequence identity was apparent with the 4-α-glucanotransferase enzymes of Streptococcus pneumoniae (46.9%), potato (42.9%) and E. coli (16.2%). The clostridial gene (designated malQ) was followed by a second ORF which, through its homology to the equivalent enzymes of E. coli and S. pneumoniae, was deduced to encode maltodextrin phosphorylase (MalP). The translation stop codon of malQ overlapped the translation start codon of the putative malP gene, suggesting that the two genes may be both transcriptionally and translationally coupled. 4-α-Glucanotransferase catalyses a disproportionation reaction in which single or multiple glucose units from oligosaccharides are transferred to the 4-hydroxyl group of acceptor sugars. Characterization of the recombinant C. butyricum enzyme demonstrated that glucose, maltose and maltotriose could act as acceptor, whereas of the three only maltotriose could act as donor. The enzyme therefore shares properties with the E. coli MalQ protein, but differs significantly from the glucanotransferase of Thermotoga maritima, which is unable to use maltotriose as donor or glucose as acceptor. Physiologically, the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase provides C. butyricum with a mechanism of utilizing amylose/maltodextrins with little drain on cellular ATP reserves.
-
-
-
Characterization of the rate-limiting step of the secretion of Bacillus subtilis α-amylase overproduced during the exponential phase of growth
More LessThe Bacillus subtilis α-amylase gene, amyE, was expressed under the regulated control of sacR, the levansucrase leader region. The gene fusion including the complete amyE coding sequence with the signal peptide sequence was integrated into the chromosome of a degU32(Hy) strain deleted of the sacB DNA fragment. In this genetic context, α-amylase is produced in the culture supernatant at a high level (2% of total protein) during the exponential phase of growth upon induction by sucrose. Pulse-chase experiments showed that the rate-limiting step (t 1/2 = 120 s) of the secretion process is the release of a cell-associated precursor form whose signal peptide has been cleaved. The efficiency of this ultimate step of secretion decreased dramatically in the presence of a metal chelator (EDTA) or when the cells were converted to protoplasts. The hypothesis that this step is tightly coupled with the folding process of α-amylase occurring within the cell wall environment was substantiated by in vitro folding studies. The unfolding-folding transition, monitored by the resistance to proteolysis, was achieved within the same time range (t 1/2 = 60 s) and required the presence of calcium. This metal requirement could possibly be satisfied in vivo by the integrity of the cell wail. The t 1/2 of the α-amylase release step is double that of levansucrase, although their folding rates are similar. This perhaps indicates that the passage through the cell wall may depend on parietal properties (e.g. metal ion binding and porosity) and on certain intrinsic properties of the protein (molecular mass and folding properties).
-
-
-
The pncA gene from naturally pyrazinamide-resistant Mycobacterium avium encodes pyrazinamidase and confers pyrazinamide susceptibility to resistant M. tuberculosis complex organisms
More LessThe antituberculosis drug pyrazinamide (PZA) needs to be converted into pyrazinoic acid (POA) by the bacterial pyrazinamidase (PZase) in order to show bactericidal activity against Mycobacterium tuberculosis. M. avium is naturally resistant to PZA. To investigate whether this natural resistance to PZA is due to inability of the M. avium PZase to convert PZA to bactericidal POA, the M. avium PZase gene (pncA) was cloned by using the M. tuberculosis pncA gene as a probe. Sequence analysis showed that the M. avium pncA gene is 561 bp long, encoding a protein with a predicted size of about 19·8 kDa; but Western blotting showed that the M. avium PZase migrated as a 24 kDa band when expressed in M. bovis BCG and Escherichia coli. Sequence comparison revealed that M. avium PZase has 67·7% and 32·8% amino acid identity with the corresponding enzymes from M. tuberculosis and E. coli, respectively. Southern blot analysis with the M. avium pncA gene as a probe showed that M. terrae, M. gastri, M. marinum, M. fortuitum, M. xenopi, M. gordonae, M. szulgai, M. celatum and M. kansasii have close pncA homologues, whereas M. chelonae and M. smegmatis did not give significant hybridization signals. Transformation with the M. avium pncA gene conferred PZA susceptibility to PZA-resistant M. tuberculosis complex organisms, indicating that the nonsusceptibility of M. avium to PZA is not due to an ineffective PZase enzyme, but appears to be related to other factors such as transport of POA.
-
-
-
Targets for pSAM2 integrase-mediated site-specific integration in the Mycobacterium smegmatis chromosome
More LessAn improved integrative cassette from plasmid pSAM2 has been constructed containing plasmid int and attP genes but excluding the xis gene, which should result in increased stability by suppression of the excision reaction. This cassette was included in both suicide and thermosensitive plasmids and used for integration in Mycobacterium smegmatis. Suicide plasmids containing this cassette integrated at a single site (attB1) in the M. smegmatis chromosome. The sequence of the attB1 site has been determined and was identified as a putative tRNAPro gene. Thermosensitive plasmids containing the cassette integrated both at the same attB1 site and at other different sites, often giving rise to simultaneous integration at two sites. A second integration site (attB2) has been sequenced, which was located in the region encoding 16S rRNA of one of the two rrn operons of M. smegmatis.
-
- Pathogenicity And Medical Microbiology
-
-
-
Comparison of biological effect of the two different enterotoxin complexes isolated from three different strains of Bacillus cerous
More LessThe cytotoxicity of the two different enterotoxin complexes of Bacillus cereus was compared after isolation from three different strains. Protein components of non-haemolytic enterotoxin (NHE) of 39 kDa, 45 kDa and 105 kDa were isolated from all of the three strains, whilst proteins B, L1 and L2 of haemolysin BL (HBL) were isolated from supernatants of two strains (F837-76 and 1230-88). These proteins were not detected in strain 0075-95. Inhibition of protein synthesis in Vero cells was used as a measure of cytotoxicity. The HBL complex from strain F837-76 was highly toxic. This strain also produced the NHE complex. However, when purified, at least two of the components of NHE had to be present in higher amounts than those of the components of HBL to cause the same degree of toxicity. Both complexes purified from strain 1230-88 were cytotoxic. The amount required to cause the same degree of cytotoxicity was approximately equal for the components of the two complexes, except that higher amounts of the 105 kDa protein of NHE had to be present than for the other components. None of the purified complexes from strain 1230-88 was toxic in amounts comparable to those of the HBL complex of strain F837-76 and NHE of strain 0075-95. These results indicate that when measuring cytotoxic enterotoxins from B. cereus at least two different complexes and six different proteins have to be taken into consideration.
-
-
-
-
Production of putative virulence factors by Renibacterium salmoninarum grown in cell culture
More LessA cell culture system, employing the fish cell line Epithelioma papillosum cyprini (EPC), was developed to study the synthesis of intracellular antigen and the expression of putative virulence factors by Renibacterium salmoninarum. EPC cultures infected with R. salmoninarum could be maintained for 7 weeks, during which the pathogen multiplied intracellularly. Immunohistochemical examination of infected cultures revealed the production of the p57 antigen, haemolysin and cytolysin. The intracellular nature of the infection was confirmed by transmission electron microscopic examination of EPC monolayers. A comparison of the relative virulence of bacterial cells cultured in EPC cells and on agar plates revealed that the former were markedly more virulent in challenge experiments with juvenile rainbow trout (Oncorhynchus mykiss Walbaum). The EPC cell culture model provided a system for the study of R. salmoninarum under more natural conditions than those achieved with plate culture techniques.
-
-
-
Evidence for Serpulina hyodysenteriae being recombinant, with an epidemic population structure
More LessThe population structure of Serpulina hyodysenteriae was investigated using multilocus enzyme electrophoresis. A total of 231 isolates were divided into 50 electrophoretic types (ETs), with a mean genetic diversity of 0·29 for the number of ETs and 0·23 for the number of isolates. Subsets of isolates from two Australian states (71 isolates from Victoria and 68 isolates from Queensland) exhibited as much genetic variation as the entire collection. The calculated index of association (I A) for the number of ETs (0·29±0·17) was not significantly different from zero, and hence provided evidence for the occurrence of significant genetic recombination accounting for the observed variation between strains. In contrast, the I A for the number of isolates (3·93.0·03) was significantly different from zero, with seven of the 50 ETs (ETs 4, 6, 13, 14, 20, 33 and 35) containing 51% of all the isolates. Even when multiple isolates from the same farm were removed from the analysis, the I A value for the number of isolates remained significantly greater than zero (I A 9·87±0·04), indicating that it was not biased by their inclusion. The results suggest that S. hyodysenteriae has an epidemic population structure.
-
- Physiology And Growth
-
-
-
Transcriptional control of several aerobically induced cytochrome structural genes in Rhodobacter sphaeroides
More LessTo decipher how the synthesis of energy-transducing enzymes responds to environmental cues, the response of three Rhodobacter sphaeroides aerobic cytochrome gene promoters was analysed under different conditions. Two of these promoters are upstream of structural genes (ctaD and coxII) for individual subunits of the cytochrome aa 3 respiratory complex. The third promoter is that for the cycFG operon, which encodes two c-type cytochromes of unknown function, cytochrome c 554 and CycG. Primer extension analysis identified a single oxygen-responsive transcription start site for each gene. Utilizing operon fusions to Escherichia coli lacZ as a measure of promoter activity, transcription from the ctaD, coxII and cycFG promoters was approximately twofold higher when cells were grown at high (30%) oxygen tensions than under low (2%) oxygen or anaerobic (photosynthetic) conditions. Analysis of promoter function using specific host mutations indicated that loss of the R. sphaeroides FNR homologue, FnrL, causes a small, but reproducible, increase in cycFG and coxII transcription when cells are grown at 2% oxygen. However, neither the ΔFnrL mutation nor alterations in sequences related to a consensus target site for the E. coli FNR protein increased function of any of these three promoters to that seen under aerobic conditions in wild-type cells. From this we conclude that FnrL is not solely responsible for reduced transcription of these three aerobic cytochrome genes under low oxygen or anaerobic conditions. When activity of these three promoters was monitored after cells were shifted from anaerobic (photosynthetic) conditions to a 30% oxygen atmosphere, it took several cell doublings for LacZ levels to increase to those found in steady-state 30% oxygen cultures. From these results, it appears that activity of these promoters is also regulated by a stable molecule whose synthesis or function responds slowly to the presence of high oxygen tensions.
-
-
-
-
Escherichia coli is unable to produce pyrroloquinoline quinone (PQQ)
More LessMany bacteria can synthesize the cofactor pyrroloquinoline quinone (PQQ), a cofactor of several dehydrogenases, including glucose dehydrogenase (GCD). Among the enteric bacteria, Klebsiella pneumoniae has been shown to contain the genes required for PQQ biosynthesis. Escherichia coli and Salmonella typhimurium were thought to be unable to synthesize PQQ but it has been reported that strain EF260, a derivative of E. coli FB8, can synthesize PQQ after mutation and can oxidize glucose to gluconate via the GCD/PQQ pathway (F. Biville, E. Turlin & F. Gasser, 1991, J Gen Microbiol 137, 1775-1782). We have reinvestigated this claim and conclude that it is most likely erroneous. (i) Strain EF260, isolated originally by Biville and coworkers, was unable to synthesize a holo-enzyme GCD unless PQQ was supplied to the growth medium. No GCD activity could be detected in membrane fractions. (ii) The amount of PQQ detected in the growth medium of EF260 was very low and not very different from that found in a medium with its parent strain or in a medium containing no cells. (iii) EF260 cells were unable to produce gluconate from glucose via the PQQ/GCD pathway. (iv) Introduction of a gcd::Cm deletion in EF260, eliminating GCD, did not affect glucose metabolism. This suggested a pathway for glucose metabolism other than the PQQ/GCD pathway, (v) Glucose uptake and metabolism in EF260 involved a low-affinity transport system of unknown identity, followed most likely by phosphorylation via glucokinase. It is concluded that E. coli cannot synthesize PQQ and that it lacks genes required for PQQ biosynthesis.
-
-
-
A novel 1,3-β-glucan synthase from the oomycete Saprolegnia monoica
An apparently novel 1,3-β-glucan synthase from the oomycete Saprolegnia monoica has been characterized. The enzyme exhibits properties that differ markedly from those of the enzyme previously described [Fèvre, M. & Dumas, C. (1977). J Gen Microbiol 103, 297-306] as it is active at alkaline pH, stimulated by the divalent cations Ca2+, Mg2+ and Mn2+, and appears to be located mainly in the apical part of the hypha. Taking into consideration the differences in pH optimum and effect of divalent ions, each enzyme activity could be assayed in the presence of the other. The insoluble polymeric product of the enzyme with alkaline pH optimum was characterized as a linear 1,3-β-glucan. Comparisons of the general properties of 1,3-β-glucan synthases suggest that enzymes from the oomycetes are more closely related to enzymes from higher plants than to those of true fungi, reflecting the fact that the oomycetes are highly divergent from chitinous fungi.
-
-
-
Polyethyleneimine is an effective permeabilizer of Gram-negative bacteria
More LessThe effect of the polycation polyethyleneimine (PEI) on the permeability properties of the Gram-negative bacterial outer membrane was investigated using Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium as target organisms. At concentrations of less than 20 µg ml−1, PEI increased the bacterial uptake of 1-N-phenylnaphthylamine, which is a hydrophobic probe whose quantum yield is greatly increased in a lipid environment, indicating increased hydrophobic permeation of the outer membrane by PEI. The effect of PEI was comparable to that brought about by the well-known permeabilizer EDTA. Permeabilization by PEI was retarded but not completely inhibited by millimolar concentrations of MgCl2. PEI also increased the susceptibility of the test species to the hydrophobic antibiotics clindamycin, erythromycin, fucidin, novobiocin and rifampicin, without being directly bactericidal. PEI sensitized the bacteria to the lytic action of the detergent SDS in assays where the bacteria were pretreated with PEI. In assays where PEI and SDS were simultaneously present, no sensitization was observed, indicating that PEI and SDS were inactivating each other. In addition, a sensitizing effect to the nonionic detergent Triton X-100 was observed for P. aeruginosa. In conclusion, PEI was shown to be a potent permeabilizer of the outer membrane of Gram-negative bacteria.
-
-
-
Mycotic acid composition of Corynebacterium glutamicum and its cell surface mutants: effects of growth with glycine and isonicotinic acid hydrazide
More LessAuxotrophic mutants of Corynebacterium glutamicum strain ATCC 13059 (parent of AS019, a rifampicin-resistant variant), which were morphologically distinct from the parent and formed protoplasts more readily, had been isolated previously. Mutants MLB130-133 and MLB194 were more sensitive to growth inhibition by isonicotinic acid hydrazide (INH) and glycine, which caused branching and budding. Fatty acid and mycolic acid (MA) profiles were determined after growth in LBG (Luria broth plus glucose), LBG-glycine (LBG- and LBG-INH (LBG-I). The fatty acid profiles of all strains were similar, except that mutant MLB133 showed some increase in stearic acid (C18:0), normally a minor component, late in the growth cycle and oleic acid proportionately decreased. All strains had five major types of MAs (C32:0, C34:0, C34:1, C36:1, C36:2) but the relative proportion of each varied with the strain, age of culture and medium composition. Mutants MLB133 and MLB194 showed slightly higher levels of non-covalentiy bound MAs than the parent and normally showed a higher proportion of longer-chained, unsaturated MAs. The proportion of extracellular MAs increased with culture age for these mutants. Typically, by late stationary phase, mycolic acids in culture fluids increased to 6.5% of the total MAs for MLB194 and 7.9% for MLB133 compared with 3.5% for the parent strain grown in LBG. The main effect of glycine (2%, w/v) addition was to increase the proportion of mycolic acids found in extracellular fluids (16.1 % for AS019 and 31% for MLB133). The most significant effects of INH were seen when strains were cultured in LBG with 8 mg INH ml−1. When harvested at late stationary phase, strains MLB133 and MLB194 had 18.8% and 21.2% extracellular mycolic acids respectively, with a significant increase in the relative proportion of unsaturated mycolic acids. This effect was not as marked for AS019, which also showed a similar decrease in C32:0 relative to increases in the proportion of C34:1 and C36:2 plus a corresponding increase in the overall proportion of unsaturated mycolic acids and increased extracellular mycolates (8.5%). These results suggest that the mutations in strains MLB133 and MLB194 are associated with synthesis of specific mycolic acids (e.g. C32:0) and attachment of mycolic acids to the cell surface, both of which are likely target sites for glycine and INH action for cell-surface modifications. In addition to previously reported targeting of the peptidoglycan cross-linking, these results show that glycine affects mycolic acid attachment to the cell surface of C. glutamicum.
-
-
-
Acetate kinase from Clostridium acetobutylicum: a highly specific enzyme that is actively transcribed during acidogenesis and solventogenesis
More LessAcetate kinase (ATP:phosphotransferase, EC 2.7.2.1) has been purified 294-fold from acid-producing cells of Clostridium acetobutylicum DSM 1731 to a specific activity of 1087 U mg−1 (ADP-forming direction). The dimeric enzyme consisted of subunits with a molecular mass of 43 kDa. The molecular mass of the native acetate kinase was in the range 87-94 kDa as judged by gel filtration and native gel electrophoresis. The enzyme showed high specificity for the substrates acetate and ATP, and maximal activity was obtained with Mn2+ as divalent cation. The presence of mercury compounds such as HgCl2 and p-hydroxymercuribenzoate resulted in an essential loss of activity. The apparent K m values for acetate, Mg-ATP, acetyl phosphate, and Mg-ADP were 73, 0.37, 0.58 and 0.71 mM. An activity-staining procedure for detection of acetate kinase in crude cell extracts after separation on native polyacrylamide gels was developed. A DNA fragment encoding 246 bp of the acetate kinase gene of C. acetobutylicum DSM 792 was cloned by a PCR-based approach. Northern blot analysis revealed transcription of the gene under acid- and solvent-producing conditions with no significant differences at the level of transcription.
-
- Genome Analysis
-
-
-
Low-resolution sequencing of Rhodobacter sphaeroides 2.A.1T: chromosome II is a true chromosome
More LessThe photosynthetic bacterium Rhodobacter sphaeroides 2.4.1T has two chromosomes, CI (∼3.0 Mb) and CII (∼0.9 Mb). In this study a low-redundancy sequencing strategy was adopted to analyse 23 out of 47 cosmids from an ordered CII library. The sum of the lengths of these 23 cosmid inserts was ∼495 kb, which comprised ∼417 kb of unique DNA. A total of 1145 sequencing runs was carried out, with each run generating 559±268 bases of sequence to give ∼640 kb of total sequence. After editing, ∼2.8% bases per run were estimated to be ambiguous. After the removal of vector and Escherichia coli sequences, the remaining ∼ 565 kb of R. sphaeroides sequences were assembled, generating ∼291 kb of unique sequences. BLASTX analysis of these unique sequences suggested that ∼131 kb (45% of the unique sequence) had matches to either known genes, or database ORFs of hypothetical or unknown function (dORFs). A total of 144 strong matches to the database was found; 101 of these matches represented genes encoding a wide variety of functions, e.g. amino acid biosynthesis, photosynthesis, nutrient transport, and various regulatory functions. Two rRNA operons (rrnB and rrnC) and five tRNAs were also identified. The remaining 160 kb of DNA sequence which did not yield database matches was then analysed using CODONPREFERENCE from the GCG package. This analysis suggested that 122 kb (42% of the total unique DNA sequence) could encode putative ORFs (ports), with the remaining 38 kb (13%) possibly representing non-coding intergenic DNA. From the data so far obtained, CII does not appear to be specialized for encoding any particular metabolic function, physiological state or growth condition. These data suggest that CII contains genes which are functionally as diverse as those found on any other bacterial chromosome and also contains sequences (pORFs) which may prove to be unique to this organism.
-
-
-
-
Sequencing of regions downstream of addA (98°) and citG (28°) in Bacillus subtilis
More LessThe nucleotide sequence of 17.3 kbp downstream of addA (98°) on the Bacillus subtilis chromosome was determined. Twenty putative ORFs were identified. Three of them coincided with known B. subtilis genes, addA, sbcD and wprA. The product of four other ORFs showed similarity to SbcC of Clostridium perfringens, CotH of B. subtilis, 2-hydroxyhepta-2,4-diene-1,7-diodate isomerase of Methanococcus jannaschi and a putative ORF of Pseudomonas syringae. In addition, a sequence of 7.6 kbp downstream of citG (189°) was analysed. Among 10 putative ORFs identified, two coincided with known genes, citG and mrgA, whilst three showed homology with X86780, a sensory protein kinase of Streptomyces hygroscopicus, an alkaline phosphatase regulatory protein and a hypothetical protease, YyxA, of B. subtilis.
-
-
-
A Bacillus subtilis chromosome segment at the 100° to 102° position encoding 11 membrane proteins
More LessThe 25·9 kbp region upstream of nprB at 100°-102° on the Bacillus subtilis chromosome was sequenced. This revealed a known gene, degA, which was previously mislocated on the genetic map. A total of 29 putative ORFs were identified including a cluster of three ORFs whose products show clear homology with sulphate adenylyl pathway enzymes and, in addition, 11 ORFs whose products have one or more membrane domains, as indicated by their hydropathy profiles.
-
-
-
The Bacillus subtilis genome from gerBC (311°) to licR (334°)
As part of the international project to sequence the Bacillus subtilis genome, the DNA region located between gerBC (311°) and licR (334°) was assigned to the Institut Pasteur. In this paper, the cloning and sequencing of 176 kb of DNA and the analysis of the sequence of the entire 271 kb region (6·5% of the B. subtilis chromosome) is described; 273 putative coding sequences were identified. Although the complete genome sequences of seven other organisms (five bacteria, one archaeon and the yeast Saccharomyces cerevisiae) are available in public databases, 65 genes from this region of the B. subtilis chromosome encode proteins without significant similarities to other known protein sequences. Among the 208 other genes, 115 have paralogues in the currently known B. subtilis DNA sequences and the products of 178 genes were found to display similarities to protein sequences from public databases for which a function is known. Classification of these genes shows a high proportion of them to be involved in the adaptation to various growth conditions (non-essential cell wall constituents, catabolic and bioenergetic pathways); a small number of the genes are essential or encode anabolic enzymes.
-
-
-
Sequence and RFLP analysis of the elongation factor Tu gene used in differentiation and classification of phytoplasmas
More LessPrimers designed from sequences of the gene encoding the elongation factor Tu (tuf gene) of several culturabie mollicutes amplified most of the tuf gene from phytoplasmas of the aster yellows, stolbur and X-disease groups. About 85% of the tuf gene from two aster yellows strains and a tomato stolbur phytoplasma was sequenced. The nucleotide sequence similarity between these related phytoplasmas was between 87·8 and 97·0%, whereas the homology with other mollicutes was 66·3-72·7%. The similarity of the deduced amino acid sequence was significantly higher, ranging from 96·0 to 99·4% among the phytoplasmas and 78·5% to 83·3% between phytoplasmas and the culturabie mollicutes examined. From the nucleotide sequences of the phytoplasma strains, two pairs of primers were designed; one amplified the phytoplasmas of most phylogenetic groups that were established, the other was specific for the aster yellows and stolbur groups. The phytoplasmas of the various groups that were amplified could be distinguished by RFLP analysis using Sau3AI, AluI and Hpall. The aster yellows group could be divided into five Sau3AI RFLP groups. These results showed that the tuf gene has the potential to be used to differentiate and classify phytoplasmas. Southern blot analysis revealed that the tuf gene is present as a single copy.
-
Volumes and issues
-
Volume 170 (2024)
-
Volume 169 (2023)
-
Volume 168 (2022)
-
Volume 167 (2021)
-
Volume 166 (2020)
-
Volume 165 (2019)
-
Volume 164 (2018)
-
Volume 163 (2017)
-
Volume 162 (2016)
-
Volume 161 (2015)
-
Volume 160 (2014)
-
Volume 159 (2013)
-
Volume 158 (2012)
-
Volume 157 (2011)
-
Volume 156 (2010)
-
Volume 155 (2009)
-
Volume 154 (2008)
-
Volume 153 (2007)
-
Volume 152 (2006)
-
Volume 151 (2005)
-
Volume 150 (2004)
-
Volume 149 (2003)
-
Volume 148 (2002)
-
Volume 147 (2001)
-
Volume 146 (2000)
-
Volume 145 (1999)
-
Volume 144 (1998)
-
Volume 143 (1997)
-
Volume 142 (1996)
-
Volume 141 (1995)
-
Volume 140 (1994)
-
Volume 139 (1993)
-
Volume 138 (1992)
-
Volume 137 (1991)
-
Volume 136 (1990)
-
Volume 135 (1989)
-
Volume 134 (1988)
-
Volume 133 (1987)
-
Volume 132 (1986)
-
Volume 131 (1985)
-
Volume 130 (1984)
-
Volume 129 (1983)
-
Volume 128 (1982)
-
Volume 127 (1981)
-
Volume 126 (1981)
-
Volume 125 (1981)
-
Volume 124 (1981)
-
Volume 123 (1981)
-
Volume 122 (1981)
-
Volume 121 (1980)
-
Volume 120 (1980)
-
Volume 119 (1980)
-
Volume 118 (1980)
-
Volume 117 (1980)
-
Volume 116 (1980)
-
Volume 115 (1979)
-
Volume 114 (1979)
-
Volume 113 (1979)
-
Volume 112 (1979)
-
Volume 111 (1979)
-
Volume 110 (1979)
-
Volume 109 (1978)
-
Volume 108 (1978)
-
Volume 107 (1978)
-
Volume 106 (1978)
-
Volume 105 (1978)
-
Volume 104 (1978)
-
Volume 103 (1977)
-
Volume 102 (1977)
-
Volume 101 (1977)
-
Volume 100 (1977)
-
Volume 99 (1977)
-
Volume 98 (1977)
-
Volume 97 (1976)
-
Volume 96 (1976)
-
Volume 95 (1976)
-
Volume 94 (1976)
-
Volume 93 (1976)
-
Volume 92 (1976)
-
Volume 91 (1975)
-
Volume 90 (1975)
-
Volume 89 (1975)
-
Volume 88 (1975)
-
Volume 87 (1975)
-
Volume 86 (1975)
-
Volume 85 (1974)
-
Volume 84 (1974)
-
Volume 83 (1974)
-
Volume 82 (1974)
-
Volume 81 (1974)
-
Volume 80 (1974)
-
Volume 79 (1973)
-
Volume 78 (1973)
-
Volume 77 (1973)
-
Volume 76 (1973)
-
Volume 75 (1973)
-
Volume 74 (1973)
-
Volume 73 (1972)
-
Volume 72 (1972)
-
Volume 71 (1972)
-
Volume 70 (1972)
-
Volume 69 (1971)
-
Volume 68 (1971)
-
Volume 67 (1971)
-
Volume 66 (1971)
-
Volume 65 (1971)
-
Volume 64 (1970)
-
Volume 63 (1970)
-
Volume 62 (1970)
-
Volume 61 (1970)
-
Volume 60 (1970)
-
Volume 59 (1969)
-
Volume 58 (1969)
-
Volume 57 (1969)
-
Volume 56 (1969)
-
Volume 55 (1969)
-
Volume 54 (1968)
-
Volume 53 (1968)
-
Volume 52 (1968)
-
Volume 51 (1968)
-
Volume 50 (1968)
-
Volume 49 (1967)
-
Volume 48 (1967)
-
Volume 47 (1967)
-
Volume 46 (1967)
-
Volume 45 (1966)
-
Volume 44 (1966)
-
Volume 43 (1966)
-
Volume 42 (1966)
-
Volume 41 (1965)
-
Volume 40 (1965)
-
Volume 39 (1965)
-
Volume 38 (1965)
-
Volume 37 (1964)
-
Volume 36 (1964)
-
Volume 35 (1964)
-
Volume 34 (1964)
-
Volume 33 (1963)
-
Volume 32 (1963)
-
Volume 31 (1963)
-
Volume 30 (1963)
-
Volume 29 (1962)
-
Volume 28 (1962)
-
Volume 27 (1962)
-
Volume 26 (1961)
-
Volume 25 (1961)
-
Volume 24 (1961)
-
Volume 23 (1960)
-
Volume 22 (1960)
-
Volume 21 (1959)
-
Volume 20 (1959)
-
Volume 19 (1958)
-
Volume 18 (1958)
-
Volume 17 (1957)
-
Volume 16 (1957)
-
Volume 15 (1956)
-
Volume 14 (1956)
-
Volume 13 (1955)
-
Volume 12 (1955)
-
Volume 11 (1954)
-
Volume 10 (1954)
-
Volume 9 (1953)
-
Volume 8 (1953)
-
Volume 7 (1952)
-
Volume 6 (1952)
-
Volume 5 (1951)
-
Volume 4 (1950)
-
Volume 3 (1949)
-
Volume 2 (1948)
-
Volume 1 (1947)