- Volume 148, Issue 4, 2002
Volume 148, Issue 4, 2002
- Review Article
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- Research Paper
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LuxS: its role in central metabolism and the in vitro synthesis of 4-hydroxy-5-methyl-3(2H)-furanone
Many bacteria produce extracellular molecules which function in cell-to-cell communication. One of these molecules, autoinducer 2 (AI-2), was first described as an extracellular signal produced by Vibrio harveyi to control luciferase expression. Subsequently, a number of bacteria have been shown to possess AI-2 activity in their culture supernatants, and bear the luxS gene product, which is required for AI-2 synthesis. In Porphyromonas gingivalis, luxS and pfs, encoding a 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH’ase), form an operon, suggesting that S-adenosylhomocysteine (SAH) or 5′-methylthioadenosine (MTA) serves as a substrate for AI-2 production. Cell-free extracts of Escherichia coli MG1655, but not DH5α (which carries a luxS frame-shift mutation) were capable of generating AI-2 activity upon addition of SAH, but not MTA. S-Ribosyl-homocysteine (RH) derived from SAH also served as a substrate in E. coli MG1655 extracts. RH-supplemented cell-free extracts of Pseudomonas aeruginosa, a bacterium that lacks luxS, only generated AI-2 activity following the introduction of a plasmid containing the Por. gingivalis pfs-luxS operon. In addition, defined in vitro systems consisting of the purified LuxS proteins from Por. gingivalis, E. coli, Neisseria meningitidis or Staphylococcus aureus converted RH to homocysteine and a compound that exhibits AI-2 activity.4-Hydroxy-5-methyl-3(2H)-furanone was identified by mass spectrometry analysis as a major product formed in this in vitro reaction. In E. coli MG1655, expression of T3SH [the bacteriophage T3 S-adenosylmethionine (SAM) hydrolase] significantly reduced AI-2 activity in culture supernatants, suggesting that AI-2 production is limited by the amount of SAH produced in SAM-dependent transmethylase reactions. The authors suggest that the LuxS protein has an important metabolic function in the recycling of SAH. They also show that Ps. aeruginosa is capable of removing AI-2 activity, implying that this molecule may act as a nutrient. In many bacteria AI-2 may in fact represent not a signal molecule but a metabolite which is released early and metabolized in the later stages of growth.
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Genetically programmed autoinducer destruction reduces virulence gene expression and swarming motility in Pseudomonas aeruginosa PAO1
The GenBank accession number for the aiiA nucleotide sequence is AF397400. The GenBank accession numbers for the nucleotide sequences of the 16S rRNA genes of strains A23 and A24 are AF397398 and AF397399.
Virulence in the opportunistic human pathogen Pseudomonas aeruginosa is controlled by cell density via diffusible signalling molecules (‘autoinducers’) of the N-acylhomoserine lactone (AHL) type. Two Bacillus sp. isolates (A23 and A24) with AHL-degrading activity were identified among a large collection of rhizosphere bacteria. From isolate A24 a gene was cloned which was similar to the aiiA gene, encoding an AHL lactonase in another Bacillus strain. Expression of the aiiA homologue from isolate A24 in P. aeruginosa PAO1 reduced the amount of the quorum sensing signal N-oxododecanoyl-L-homoserine lactone and completely prevented the accumulation of the second AHL signal, N-butyryl-L-homoserine lactone. This strongly reduced AHL content correlated with a markedly decreased expression and production of several virulence factors and cytotoxic compounds such as elastase, rhamnolipids, hydrogen cyanide and pyocyanin, and strongly reduced swarming. However, no effect was observed on flagellar swimming or on twitching motility, and aiiA expression did not affect bacterial adhesion to a polyvinylchloride surface. In conclusion, introduction of an AHL degradation gene into P. aeruginosa could block cell–cell communication and exoproduct formation, but failed to interfere with surface colonization.
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Potassium- or sodium-efflux ATPase, a key enzyme in the evolution of fungi
More LessThe GenBank accession numbers for the sequences reported in this paper are: Pleurotus ostreatus ENA1, AJ420741; Phycomyces blakesleeanus ENA1, AJ420742; Ph. blakesleeanus PCA1, AJ420743; Blakeslea trispora ENA1, AJ420744; B. trispora BCA1, AJ420745; B. trispora BCA2, AJ420746.
Potassium is the most abundant cation in cells. Therefore, plant-associated fungi and intracellular parasites are permanently or circumstantially exposed to high K+ and must avoid excessive K+ accumulation activating K+ efflux systems. Because high K+ and high pH are compatible in natural environments, free-living organisms cannot keep a permanent transmembrane ΔpH and cannot rely only on K+/H+ antiporters, as do mitochondria. This study shows that the Schizosaccharomyces pombe CTA3 is a K+-efflux ATPase, and that other fungi are furnished with Na+-efflux ATPases, which also pump Na+. All these fungal ATPases, including those pumping only Na+, form a phylogenetic group, IID or ENA, among P-type ATPases. By searching in databases and partial cloning of ENA genes in species of Zygomycetes and Basidiomycetes, the authors conclude that probably all fungi have these genes. This study indicates that fungal K+- or Na+-ATPases evolved from an ancestral K+-ATPase, through processes of gene duplication. In yeast hemiascomycetes these duplications have occurred recently and produced bifunctional ATPases, whereas in Neurospora, and probably in other euascomycetes, they occurred earlier in evolution and produced specialized ATPases. In Schizosaccharomyces, adaptation to Na+ did not involve the duplication of the K+-ATPase and thus it retains an enzyme which is probably close to the original one. The parasites Leishmania and Trypanosoma have ATPases phylogenetically related to fungal K+-ATPases, which are probably functional homologues of the fungal enzymes.
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Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria
More LessThe inability to transform many clinically important Gram-negative bacteria has hampered genetic studies addressing the mechanism of bacterial pathogenesis. This report describes the development and construction of a delivery system utilizing the broad-host-range transducing bacteriophage P1. The phagemids used in this system contain a P1 pac initiation site to package the vector, a P1 lytic replicon to generate concatemeric DNA, a broad-host-range origin of replication and an antibiotic-resistance determinant to select bacterial clones containing the recircularized phagemid. Phagemid DNA was successfully introduced by infection and stably maintained in members of the families Enterobacteriaceae (Escherichia coli, Shigella flexneri, Shigella dysenteriae, Klebsiella pneumoniae and Citrobacter freundii) and Pseudomonadaceae (Pseudomonas aeruginosa). In addition to laboratory strains, these virions were used successfully to deliver phagemids to a number of strains isolated from patients. This ability to deliver genetic information to wild-type strains raises the potential for use in antimicrobial therapies and DNA vaccine development.
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MabA (FabG1), a Mycobacterium tuberculosis protein involved in the long-chain fatty acid elongation system FAS-II
The fatty acid elongation system FAS-II is involved in the biosynthesis of mycolic acids, which are very long-chain fatty acids of the cell envelope specific to Mycobacterium tuberculosis and other mycobacteria. A potential component of FAS-II, the protein MabA (FabG1), was overexpressed and purified. Sedimentation equilibrium analyses revealed that MabA undergoes a dimer to tetramer self-association with a dissociation constant of 22 μM. The protein was detected by Western blotting in a mycobacterial cell-wall extract that produces mycolic acids and in the FPLC FAS-II fraction. MabA was shown to catalyse the NADPH-specific reduction of β-ketoacyl derivatives, equivalent to the second step of a FAS-II elongation round. Unlike the known homologous proteins, MabA preferentially metabolizes long-chain substrates (C8–C20) and has a poor affinity for the C4 substrate, in agreement with FAS-II specificities. Molecular modelling of MabA structure suggested the presence of an unusually hydrophobic substrate-binding pocket holding a unique Trp residue, suitable for fluorescence spectroscopic analyses. In agreement with the enzyme kinetic data, the spectral properties of MabA were different in the presence of the C8–C16 ligands as compared to the C4 ligand. Altogether, these data bring out distinctive enzymic and structural properties of MabA, which correlate with its predilection for long-chain substrates, in contrast to most of the other known ketoacyl reductases.
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Physiological consequences associated with overproduction of Mycobacterium tuberculosis FtsZ in mycobacterial hosts
The ftsZ gene of Mycobacterium tuberculosis H37Rv has been characterized as the first step in determining the molecular events involved in the cell division process in mycobacteria. Western analysis revealed that intracellular levels of FtsZ are growth phase dependent in both M. tuberculosis and Mycobacterium smegmatis. Unregulated expression of M. tuberculosis ftsZ from constitutive hsp60 and dnaA promoters in M. tuberculosis hosts resulted in lethality whereas expression from only the hsp60 promoter was toxic in M. smegmatis hosts. Expression of ftsZ from the dnaA promoter in M. smegmatis resulted in ∼sixfold overproduction and the merodiploids exhibited slow growth, an increased tendency to clump and filament, and in some cases produced buds and branches. Many of the cells also contained abnormal and multiple septa. Expression of ftsZ from the chemically inducible acetamidase promoter in M. smegmatis hosts resulted in ∼22-fold overproduction of FtsZ and produced filamentous cells, many of which lacked any visible septa. Visualization of the M. tuberculosis FtsZ tagged with green fluorescent protein in M. smegmatis by fluorescence microscopy revealed multiple fluorescent FtsZ foci, suggesting that steps subsequent to the formation of organized FtsZ structures but prior to septum formation are blocked in FtsZ-overproducing cells. Together these results suggest that the intracellular concentration of FtsZ protein is critical for productive septum formation in mycobacteria.
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Characterization of the genetic locus responsible for the production of ABP-118, a novel bacteriocin produced by the probiotic bacterium Lactobacillus salivarius subsp. salivarius UCC118
The GenBank accession number for the sequence reported in this paper is AF408405.
ABP-118, a small heat-stable bacteriocin produced by Lactobacillus salivarius subsp. salivarius UCC118, a strain isolated from the ileal–caecal region of the human gastrointestinal tract, was purified to homogeneity. Using reverse genetics, a DNA fragment specifying part of ABP-118 was identified on a 10769 bp chromosomal region. Analysis of this region revealed that ABP-118 was a Class IIb two-peptide bacteriocin composed of Abp118α, which exhibited the antimicrobial activity, and Abp118β, which enhanced the antimicrobial activity. The gene conferring strain UCC118 immunity to the action of ABP-118, abpIM, was identified downstream of the abp118β gene. Located further downstream of abp118β, several ORFs were identified whose deduced proteins resembled those of proteins involved in bacteriocin regulation and secretion. Heterologous expression of ABP-118 was achieved in Lactobacillus plantarum, Lactococcus lactis and Bacillus cereus. In addition, the abp118 locus encoded an inducing peptide, AbpIP, which was shown to play a role in the regulation of ABP-118 production. This novel bacteriocin is, to the authors’ knowledge, the first to be isolated from a known human probiotic bacterium and to be characterized at the genetic level.
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Sequence analysis of the lactococcal bacteriophage bIL170: insights into structural proteins and HNH endonucleases in dairy phages
More LessThe GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are bIL170, AF009630; bIL120, AY054975; bIL15, AY054976; bIL191, AY054977; bIL77, AY054978.
The complete 31754 bp genome of bIL170, a virulent bacteriophage of Lactococcus lactis belonging to the 936 group, was analysed. Sixty-four ORFs were predicted and the function of 16 of them was assigned by significant homology to proteins in databases. Three putative homing endonucleases of the HNH family were found in the early region. An HNH endonuclease with zinc-binding motif was identified in the late cluster, potentially being part of the same functional module as terminase. Three putative structural proteins were analysed in detail and show interesting features among dairy phages. Notably, gpl12 (putative fibre) and gpl20 (putative baseplate protein) of bIL170 are related by at least one of their domains to a number of multi-domain proteins encoded by lactococcal or streptococcal phages. A 110- to 150-aa-long hypervariable domain flanked by two conserved motifs of about 20 aa was identified. The analysis presented here supports the participation of some of these proteins in host-range determination and suggests that specific adsorption to the host may involve a complex multi-component system. Divergences in the genome of phages of the 936 group, that may have important biological properties, were noted. Insertions/deletions of units of one or two ORFs were the main source of divergence in the early clusters of the two entirely sequenced phages, bIL170 and sk1. An exchange of fragments probably affected the regions containing the putative origin of replication. It led to the absence in bIL170 of the direct repeats recognized in sk1 and to the presence of different ORFs in the ori region. Shuffling of protein domains affected the endolysin (putative cell-wall binding part), as well as gpl12 and gpl20.
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Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis
The GenBank accession number for the sequence reported in this paper is AY046926.
Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches ’bottle-necks’ are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected ’rate-limiting’ steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates.
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Expression of the Escherichia coli yfiD gene responds to intracellular pH and reduces the accumulation of acidic metabolic end products
The YfiD protein of Escherichia coli has been reported to be an acid-inducible protein. Here it is shown that expression of a yfiD::lac reporter fusion is enhanced up to 3·5-fold during acidic growth. The anaerobic transcription factor FNR was confirmed as the major regulator of yfiD expression, and ArcA was found to enhance anaerobic yfiD expression, probably by displacing a repressing FNR dimer in the −93·5 region of the promoter. Moreover, the pyruvate sensor PdhR was shown to act as a minor anaerobic repressor of yfiD expression. On the basis of its strong homology to the C-terminal region of pyruvate formate-lyase (PFL) it was predicted that YfiD would be a radical-containing enzyme. The YfiD radical was found to be introduced by the PFL-activase enzyme, but unlike PFL, AdhE did not deactivate radicalized YfiD. The extent of radical activation of YfiD was enhanced by low intracellular pH, and thus it was concluded that both yfiD expression and YfiD activity are affected by growth at low pH. The yfiD mutant strain JRG4033 excreted increased levels of organic acids compared to the parental strain when grown in chemostat culture under oxygen-starved conditions, consistent with the acid-inducibility of yfiD expression and the recently reported ability of YfiD to rescue the activity of oxygenolytically cleaved PFL.
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Escherichia coli aconitases and oxidative stress: post-transcriptional regulation of sodA expression
More LessEscherichia coli possesses two aconitases, a stationary-phase enzyme (AcnA), which is induced by iron and oxidative stress, and a major but less stable enzyme (AcnB), synthesized during exponential growth. In addition to the catalytic activities of the holo-proteins, the apo-proteins function as post-transcriptional regulators by site-specific binding to acn mRNAs. Thus, it has been suggested that inactivation of the enzymes could mediate a rapidly reacting post-transcriptional component of the bacterial oxidative stress response. Here it is shown that E. coli acn mutants are hypersensitive to the redox-stress reagents H2O2 and methyl viologen. Proteomic analyses further revealed that the level of superoxide dismutase (SodA) is enhanced in acnB and acnAB mutants, and by exposure to methyl viologen. The amounts of other proteins, including thioredoxin reductase, 2-oxoglutarate dehydrogenase, succinyl-CoA synthetase and chaperone proteins, were also affected in the acn mutants. The altered patterns of sodA expression were confirmed in studies with sodA–lacZ reporter strains. Quantitative Northern blotting indicated that AcnA enhances the stability of the sodA transcript, whereas AcnB lowers its stability. Direct evidence that the apo-proteins have positive (AcnA) and negative (AcnB) effects on SodA synthesis was obtained from in vitro transcription–translation experiments. It is suggested that the aconitase proteins of E. coli serve as a protective buffer against the basal level of oxidative stress that accompanies aerobic growth by acting as a sink for reactive oxygen species and by modulating translation of the sodA transcript.
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Intracellular cyclic AMP concentration is decreased in Salmonella typhimurium fur mutants
More LessThe GenBank accession number for the sequence reported in this paper is AF268282.
It is known that the Fur protein negatively regulates iron-uptake systems in different bacterial species, including Salmonella typhimurium. In this study it has been shown that the intracellular concentration of cyclic AMP (cAMP) is lower in a knockout S. typhimurium fur mutant than in the wild-type strain. According to this, the expression of two cAMP-regulated genes, such as pepE (encoding an α-aspartyl dipeptidase) and the Escherichia coli lac operon, is decreased in S. typhimurium fur cells in comparison with wild-type cells. Introduction of an additional mutation in cpdA, encoding a cyclic 3′,5′-cAMP phosphodiesterase, recovers wild-type intracellular cAMP concentration in the S. typhimurium fur mutant. Likewise, expression of pepE and the E. coli lac operon was the same in the S. typhimurium fur cpdA double mutant and the wild-type strain. Moreover, these results also demonstrate that the S. typhimurium Fur protein positively regulates the expression of the flhD master operon governing the flagellar regulon. This positive control must be mediated by binding of the S. typhimurium Fur protein to the flhD promoter as indicated by the fact that this promoter tests positive in a Fur titration assay.
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The Candida albicans Sup35p protein (CaSup35p): function, prion-like behaviour and an associated polyglutamine length polymorphism
The GenBank accession numbers for the sequences determined in this work are AF020554 (CaSUP35 gene) and AY028660–AY028673 (CaSUP35 N domains).
The Sup35p protein of Saccharomyces cerevisiae is an essential translation factor whose prion-like properties give rise to the non-Mendelian genetic element [PSI +]. In this study the SUP35 gene from the related yeast species Candida albicans has been characterized. The CaSUP35 gene encodes a protein (CaSup35p) of 729 aa which shows 65% amino acid identity to the S. cerevisiae Sup35p protein (ScSup35p), with the C-terminal region showing greater identity (79%) than the N-terminal region. The full-length CaSup35p can functionally replace ScSup35p in S. cerevisiae although complementation is only complete when CaSup35p is overexpressed. Complementation only requires expression of the CaSup35p C domain. In S. cerevisiae the full-length CaSup35p is unable to establish a prion-like aggregated state even in the presence of endogenous ScSup35p prion ‘seeds’, thus confirming the existence of a species barrier in fungal prion propagation. Subcellular localization studies in C. albicans show that although CaSup35p is normally ribosome-associated, when not ribosome-associated, it does not form pelletable high-molecular-mass aggregates characteristic of the ScSup35p in [PSI +] strains. Unlike the ScSup35p, the CaSup35p N domain contains a number of polyglutamine repeats although it does contain seven copies of the peptide GGYQQ that is repeated in the ScSup35p N domain. Analysis of the CaSUP35 gene from 14 different strains of C. albicans identified four naturally occurring polymorphisms associated with changes in the length of the largest of the polyglutamine repeats. These findings have important implications for the evolution of fungal prion genes.
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Homozygosity at the Candida albicans MTL locus associated with azole resistance
More LessAntifungal drug resistance in the pathogenic fungus Candida albicans is a serious threat to the growing population of immunocompromised patients. This study describes a significant correlation between loss of heterozygosity at the C. albicans mating-type-like (MTL) locus and resistance to azole antifungals. A pool of 96 clinical isolates consisting of 50 azole-resistant or susceptible dose-dependent isolates and 46 azole-susceptible isolates was screened by PCR for the presence of MTLa1 and MTLα1. These genes were used as markers for the MTLa and MTLα loci. Both loci were present in 84 of the isolates. Six isolates failed to amplify MTLa1 and six failed to amplify MTLα1. Further PCR analysis demonstrated that loss of the MTLa1 and MTLα1 genes corresponded to loss of all of the loci-specific genes, resulting in homozygosity at the MTL locus. Southern analysis and single nucleotide polymorphism (SNP) analysis were used to determine that this loss of heterogeneity was due to replacement of one of the MTL loci with a duplicate of the other locus resulting in two homozygous copies of the MTL locus. Of the 12 homozygous isolates, one isolate was sensitive to azole drugs. Statistical analysis of the data demonstrates a strong correlation between homozygosity at the MTL locus and azole resistance (P<0·003). In a set of serial isolates, an increase in azole resistance correlated with the loss of heterozygosity at the MTL locus, lending further strength to the correlation. Gene disruptions of the MTL loci were found to have no effect on azole susceptibility.
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Complications in cell-surface labelling by biotinylation of Candida albicans due to avidin conjugate binding to cell-wall proteins
More LessInitial contact between the opportunistic fungal pathogen Candida albicans and host tissue occurs at the cell surface. Biotin derivatives have been used to label the cell-surface proteins of yeasts, with labelled proteins subsequently detected by avidin–reporter conjugates. Previous work has indicated that avidin can bind to C. albicans proteins in the absence of biotin, suggesting a possible host-cell-recognition mechanism by fungal cell-surface proteins. To investigate this mechanism, Western blots of proteins extracted from biotinylated and mock-treated cells were probed with avidin or modified-avidin reagents. Each avidin reagent bound to cell-wall proteins extracted from non-biotinylated cells. Binding did not appear to be due to the lectin-like activity of the cell-wall proteins of C. albicans or to the presence of biotin in the sample itself. Binding was inhibited by added biotin, by the chaotrope KSCN and by NaCl in a concentration-dependent manner, although inhibition varied among the avidin conjugates tested. Thus, the non-specific binding of avidin to the cell-wall proteins of C. albicans appears to involve hydrophobic and electrostatic interactions, depending on the particular avidin species. These observations demonstrate potential pitfalls in the use of avidin–biotin complexes to identify cell-surface molecules and could provide insights into protein–protein interactions at the C. albicans cell wall.
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Identification of a role for Saccharomyces cerevisiae Cgr1p in pre-rRNA processing and 60S ribosome subunit synthesis
More LessSaccharomyces cerevisiae CGR1 encodes a conserved fungal protein that localizes to the nucleolus. To determine if this localization reflects a role for Cgr1p in ribosome biogenesis two yeast cgr1 mutants were examined for defects in ribosome synthesis: a conditional depletion strain in which CGR1 is under the control of a tetracycline-repressible promoter and a mutant strain in which a C-terminal truncated Cgr1p is expressed. Both strains had impaired growth rates and were hypersensitive to the aminoglycosides paromomycin and hygromycin. Polysome analyses of the mutants revealed increased levels of free 40S subunits relative to 60S subunits, a decrease in 80S monosomes and accumulation of half-mer polysomes. Pulse–chase labelling demonstrated that pre-rRNA processing was defective in the mutants, resulting in accumulation of the 35S, 27S and 7S pre-rRNAs and delayed production of the mature 25S and 5·8S rRNAs. The synthesis of the 18S and 5S rRNAs was unaffected. Loss of Cgr1 function also caused a partial delocalization of the 5′-ITS1 RNA and the nucleolar protein Nop1p into the nucleoplasm, suggesting that Cgr1p contributes to compartmentalization of nucleolar constituents. Together these findings establish a role for Cgr1p in ribosome biogenesis.
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Biosynthesis of the dideoxysugar component of jadomycin B: genes in the jad cluster of Streptomyces venezuelae ISP5230 for l-digitoxose assembly and transfer to the angucycline aglycone
More LessThe GenBank accession number for the sequence reported in this paper is AY026363.
Eight additional genes, jadX, O, P, Q, S, T, U and V, in the jad cluster of Streptomyces venezuelae ISP5230, were located immediately downstream of jadN by chromosome walking. Sequence analyses and comparisons implicated them in biosynthesis of the 2,6-dideoxysugar in jadomycin B. The genes were cloned in Escherichia coli, inactivated by inserting an apramycin resistance cassette with a promoter driving transcription of downstream genes, and transferred into Streptomyces venezuelae by intergeneric conjugation. Analysis by HPLC and NMR of intermediates accumulated by cultures of the insertionally inactivated Streptomyces venezuelae mutants indicated that jadO, P, Q, S, T, U and V mediate formation of the dideoxysugar moiety of jadomycin B and its attachment to the aglycone. Based on these results and sequence similarities to genes described in other species producing deoxysugar derivatives, a biosynthetic pathway is proposed in which the jadQ product (glucose-1-phosphate nucleotidyltransferase) activates glucose to its nucleotide diphosphate (NDP) derivative, and the jadT product (a 4,6-dehydratase) converts this to NDP-4-keto-6-deoxy-D-glucose. An NDP-hexose 2,3-dehydratase and an oxidoreductase, encoded by jadO and jadP, respectively, catalyse ensuing reactions that produce an NDP-2,6-dideoxy-D-threo-4-hexulose. The product of jadU (NDP-4-keto-2,6-dideoxy-5-epimerase) converts this intermediate to its L-erythro form and the jadV product (NDP-4-keto-2,6-dideoxyhexose 4-ketoreductase) reduces the keto group of the NDP-4-hexulose to give an activated form of the L-digitoxose moiety in jadomycin B. Finally, a glycosyltransferase encoded by jadS transfers the activated sugar to jadomycin aglycone. The function of jadX is unclear; the gene is not essential for jadomycin B biosynthesis, but its presence ensures complete conversion of the aglycone to the glycoside. The deduced amino acid sequence of a 612 bp ORF (jadR*) downstream of the dideoxysugar biosynthesis genes resembles many TetR-family transcriptional regulator sequences.
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Nonribosomal biosynthesis of vancomycin-type antibiotics: a heptapeptide backbone and eight peptide synthetase modules
The GenBank/EMBL/DDBJ accession number for the balhimycin biosynthetic gene sequence reported in this paper is Y16952.
During analysis of the recently identified gene cluster for the glycopeptide antibiotic balhimycin, produced by Amycolatopsis mediterranei DSM 5908, novel genes were identified and characterized in detail. The gene products of four of the identified genes (bpsA, bpsB, bpsC and bpsD) are nonribosomal peptide synthetases (NRPSs); one (Orf1-protein) shows similarities to small proteins associated with several NRPSs without an assigned function. BpsA and BpsB are composed of three modules each (modules 1–6), BpsC of one module (module 7) and BpsD of a minimal module (module 8). Thus, the balhimycin gene cluster encodes eight modules, whereas its biosynthetic product is a heptapeptide. Non-producing mutants were created by a gene disruption of bpsB, an in-frame deletion of bpsC and a gene replacement of bpsD. After establishment of a gene complementation system for Amycolatopsis strains, the replacement mutant of bpsD was complemented, demonstrating for the first time that BpsD, encoding the eighth module, is indeed involved in balhimycin biosynthesis. After feeding with β-hydroxytyrosine the capability of the bpsD mutant to produce balhimycin was restored, demonstrating the participation of BpsD in the biosynthesis of this amino acid. The specificity of four of the eight adenylation domains was determined by ATP/PPi exchange assays: modules 4 and 5 activated L-4-hydroxyphenylglycine, module 6 activated β-hydroxytyrosine and module 7 activated L-3,5-dihydroxyphenylglycine, which is in accordance with the sequence of the non-proteogenic amino acids 4 to 7 of the balhimycin backbone.
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Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover
N-acyl-L-homoserine lactones (AHLs) are co-regulatory ligands required for control of the expression of genes encoding virulence traits in many Gram-negative bacterial species. Recent studies have indicated that AHLs modulate the cellular concentrations of LuxR-type regulatory proteins by binding and fortifying these proteins against proteolytic degradation (Zhu & Winans, 2001 R43 ). Halogenated furanones produced by the macroalga Delisea pulchra inhibit AHL-dependent gene expression. This study assayed for an in vivo interaction between a tritiated halogenated furanone and the LuxR protein of Vibrio fischeri overproduced in Escherichia coli. Whilst a stable interaction between the algal metabolite and the bacterial protein was not found, it was noted by Western analysis that the half-life of the protein is reduced up to 100-fold in the presence of halogenated furanones. This suggests that halogenated furanones modulate LuxR activity but act to destabilize, rather than protect, the AHL-dependent transcriptional activator. The furanone-dependent reduction in the cellular concentration of the LuxR protein was associated with a reduction in expression of a plasmid encoded P luxI –gfp(ASV) fusion suggesting that the reduction in LuxR concentration is the mechanism by which furanones control expression of AHL-dependent phenotypes. The mode of action by which halogenated furanones reduce cellular concentrations of the LuxR protein remains to be characterized.
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Redundancy, phylogeny and differential expression of Histoplasma capsulatum catalases
More LessThe GenBank accession numbers for the cDNA sequences reported in this paper are AF139985 (CATB), AF189368 (CATA) and AF189369 (CATP).
Histoplasma capsulatum produces an extracellular catalase termed M antigen, which is similar to catalase B of Aspergillus and Emericella species. Evidence is presented here for two additional catalase isozymes in H. capsulatum. Catalase A is highly similar to a large-subunit catalase in Aspergillus and Emericella species, while catalase P is a small-subunit catalase protein with greatest similarity to known peroxisomal catalases of animals and Saccharomycotina yeasts. Complete cDNAs for the CATA and CATP genes (encoding catalases A and P, respectively) were isolated. The transcriptional expression of the H. capsulatum CATA, CATB (M antigen) and CATP genes was assessed by Northern blot hybridizations on total RNA. Results at the transcript levels for these genes are shown for three conditions: cell morphology (mycelial versus yeast phase cells), oxidative stress (in response to a challenge with H2O2) and carbon source (glucose vs glycerol). Collectively, these results demonstrated regulation of CATA by both cell morphology and oxidative stress, but not by carbon source, and regulation of CATB and CATP by carbon source but not cell morphology or oxidative stress. A phylogenetic analysis of presently available catalase sequences and intron residences was done. The results support a model for evolution of eukaryotic monofunctional catalase genes from prokaryotic genes.
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Efflux of organic acids in Penicillium simplicissimum is an energy-spilling process, adjusting the catabolic carbon flow to the nutrient supply and the activity of catabolic pathways
More LessContinuous cultivation was used to study the effect of glucose, ammonium, nitrate or phosphate limitation on the excretion of tricarboxylic acid (TCA) cycle intermediates by Penicillium simplicissimum. Additionally, the effect of benzoic acid, salicylhydroxamic acid (SHAM) and 2,4-dinitrophenol on TCA cycle intermediates was studied. The physiological state of the fungus was characterized by its glucose and O2 consumption, its CO2 production, its intra- and extracellular concentrations of TCA cycle intermediates, as well as by its biomass yield, its maintenance coefficient and its respiratory quotient. The excretion of TCA cycle intermediates was observed during ammonium-, nitrate- and phosphate-limited growth. The highest productivity was found with phosphate-limited growth. The respiratory quotient was 1·3 under ammonium limitation and 0·7 under phosphate limitation. Citrate was always the main excreted intermediate. This justifies calling this excretion an energy-spilling process, because citrate excretion avoids the synthesis of too much NADH. The addition of benzoic acid further increased the excretion of TCA cycle intermediates by ammonium-limited hyphae. A SHAM-sensitive respiration was constitutively present during ammonium-limited growth of the fungus. The sum of the excreted organic acids was negatively correlated with the biomass yield (Y GlcX).
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Isolation and biochemical characterization of an endo-1,3-β-glucanase from Streptomyces sioyaensis containing a C-terminal family 6 carbohydrate-binding module that binds to 1,3-β-glucan
More LessThe GenBank accession number for the sequence reported in this paper is AF21741.
A gene encoding 1,3-β-glucanase was isolated from Streptomyces sioyaensis based on an activity plate assay. Analysis of the deduced amino acid sequence of the gene revealed that the matured 1,3-β-glucanase has two functional domains separated by a stretch of nine glycine residues. The N-terminal domain shares sequence similarity with bacterial endo-1,3-β-glucanases classified in glycosyl hydrolase family 16 (GHF 16), while the C-terminal domain is a putative carbohydrate-binding module (CBM) grouped into CBM family 6. To characterize the function of each domain, both the full-length and the CBM-truncated versions of the protein were expressed in Escherichia coli and purified to homogeneity. Biochemical data suggest that the glycosyl hydrolase domain preferentially catalyses the hydrolysis of glucans with 1,3-β linkage, and has an endolytic mode of action. Binding assay indicated that the C-terminal CBM binds to various insoluble β-glucans (1,3-, 1,3–1,4- and 1,4- linkages) but not to xylan, a primary binding target for most members of CBM family 6. The full-length and the CBM-truncated proteins had similar specific activity (units per mol of hydrolase domain) on soluble 1,3-β-glucan, whereas the former had much stronger specific activity on insoluble 1,3-β-glucans, suggesting that the C-terminal CBM enhances the activity of the S. sioyaensis 1,3-β-glucanase against insoluble substrates, presumably by increasing the frequency of encounter events between the hydrolase domain and the substrate.
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Influence of extracellular polymeric substances on deposition and redeposition of Pseudomonas aeruginosa to surfaces
In this study, the role of extracellular polymeric substances (EPS) in the initial adhesion of EPS-producing Pseudomonas aeruginosa SG81 and SG81R1, a non-EPS-producing strain, to substrata with different hydrophobicity was investigated. The release of EPS by SG81 was concurrent with a decrease in surface tension of a bacterial suspension from 70 to 45 mJ m−2 that was absent for SG81R1. Both strains adhered faster and in higher numbers to a hydrophilic than to a hydrophobic substratum, but the initial deposition rates and numbers of adhering bacteria in a stationary-end point were highest for the non-EPS-producing strain SG81R1, regardless of substratum hydrophobicity. Both strains adhered less to substrata pre-coated with isolated EPS of strain SG81. Furthermore, it was investigated whether bacteria, detached by passing air-bubbles, had left behind ‘footprints’ with an influence on adhesion of newly redepositing bacteria. Redeposition on glass was highest for non-EPS-producing SG81R1 and decreased linearly with the number of times these cycles of detachment and deposition were repeated to become similar to the redeposition of SG81 after six cycles. This indicates that P. aeruginosa SG81 leaves the substratum surface nearly completely covered with EPS after detachment, while SG81R1 releases only minor amounts of surface active EPS, completely covering the substratum after repeated cycles of detachment and adhesion. Atomic force microscopy showed a thick and irregular EPS layer (up to 32 nm) after the first detachment cycle of EPS-producing strain SG81, whereas the putatively non-EPS-producing strain SG81R1 left a 9 nm thin layer after one cycle. X-ray photoelectron spectroscopy indicated that the bacterial footprints consisted of uronic acids, the prevalence of which increased with the number of detachment and deposition cycles.
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Envelope instability in DNA adenine methylase mutants of Salmonella enterica
More LessMutants of Salmonella enterica serovar Typhimurium lacking DNA adenine (Dam) methylase show reduced secretion of invasion effectors encoded in the Salmonella-pathogenicity island 1 (SPI-1). Concomitant with this alteration, a high number and quantity of extracellular proteins are detected in cultures of Dam− mutants. This study shows by subcellular fractionation analysis that the presence of numerous extracellular proteins in cultures of Dam− mutants is linked to an exacerbated release of membrane particulate material. The membrane ‘leaky’ phenotype and the impaired functionality of type III secretion systems were, however, unrelated since exacerbated release of proteins to the medium was evident in Dam− strains carrying mutations in either SPI-1 (invA, invJ) or flagellar (flhD) genes. This result supports the view that Dam methylation controls a plethora of cellular processes. Electron microscopy analysis demonstrated that the accumulation of membrane particulate material occurs preferentially as vesicles in stationary cultures of Dam− strains. In addition, a reduction in the relative amount of peptidoglycan-associated lipoprotein (PAL), TolB, OmpA and murein lipoprotein (Lpp) bound to peptidoglycan was observed in actively growing Dam− mutants. The existence of an envelope defect was further confirmed by the increased sensitivity to deoxycholate exhibited by Dam− mutants, mostly during exponential growth. Unexpectedly, lack of Dam methylation neither increased envelope instability nor impaired the association of PAL-Tol-Lpp proteins to the peptidoglycan in Escherichia coli. Accordingly, E. coli Dam− mutants did not show sensitivity to deoxycholate. Altogether, these results indicate that, besides its role in modulating the secretion of effectors by the SPI-1-encoded type III apparatus, Dam methylation controls cell envelope integrity in S. enterica.
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Construction and characterization of a nonpigmented mutant of Porphyromonas gingivalis: cell surface polysaccharide as an anchorage for gingipains
The GenBank/EMBL/DDBJ accession number for the sequences reported in this paper is D64132.
A nonpigmented mutant of Porphyromonas gingivalis was constructed by using transposon mutagenesis. The mutant possessed the transposon DNA at the novel gene porR. Gene targeted mutagenesis revealed that porR was responsible for pigmentation. The porR gene shared similarities with genes of the degT family, the products of which are now considered to be transaminases involved in biosynthesis of sugar portions of cell-surface polysaccharides and aminoglycosides. The porR mutant showed a pleiotropic phenotype: delayed maturation of fimbrillin, preferential presence of Rgp and Kgp proteinases in culture supernatants, and no haemagglutination. The porR mutant had altered phenol extractable polysaccharide compared to the porR + sibling strain. A mAb, 1B5, that reacts with sugar portions of P. gingivalis cell surface polysaccharide and membrane-type Rgp proteinase showed no reaction with the cell lysates of the porR mutant. These results indicate that porR is involved in biosynthesis of cell surface polysaccharide that may function as an anchorage for Rgp, Kgp, haemagglutinins and the haemoglobin receptor protein.
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Interaction of human Tamm–Horsfall glycoprotein with Bordetella pertussis toxin
Tamm–Horsfall glycoprotein (THP), which is synthesized by renal tubular cells, is the most abundant protein in normal human urine. Although its physiological function remains unclear, it has been proposed that THP may act as a defence factor against urinary tract infections by inhibiting the binding of S- and P-fimbriated Escherichia coli to renal epithelial cells. Because THP-related proteins are also found in the superficial layers of the oral mucosa, the authors investigated the ability of THP to interfere with the cytoadherence of pathogenic bacteria that colonize mucosal surfaces other than those of the urogenital tract. In this report, it is shown that THP binds to virulent Bordetella pertussis and reduces its adherence to both renal and pulmonary epithelial cells. This cytoadherence inhibitory effect was not observed with a B. pertussis mutant lacking the pertussis toxin (PTX) operon, and was dependent on the direct interaction of THP with the S2 subunit within the PTX B oligomer. The authors also show that the glycosylation moiety of THP is crucial for its binding to PTX. The THP–PTX interaction was exploited to develop an affinity chromatography method that allows a one-step purification of active PTX. These observations suggest that besides its anti-adherence activity, THP may also trap toxins produced by pathogenic bacteria that colonize mucosal surfaces.
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Identification of genetic differences between two Campylobacter jejuni strains with different colonization potentials
More LessThe GenBank accession numbers for the sequences reported in this paper can be found in Table 2.
The consumption of poultry meat contaminated with Campylobacter jejuni is considered to be a risk factor for human campylobacteriosis. The development of targeted strategies to control campylobacters in broilers would benefit from knowledge of those bacterial factors important in colonization of the avian gut. During preliminary studies it was noted that C. jejuni NCTC 11168 was a poorer colonizer of chickens than strain 81116. This poor colonization could not be fully restored by in vivo passage, suggesting that it was a genetically endowed property of strain 11168. As the genome sequence is available for this strain, the technique of subtractive hybridization was used to identify gene fragments of strain 81116 not present in strain 11168. After two screening cycles, 24 out of 42 clones were identified as having DNA inserts specific for strain 81116. Six of these 24 clones contained gene fragment inserts with similarities to restriction–modification enzymes found in other bacteria. Two inserts had similarity to arsenic-resistance genes, whereas four others had similarities to cytochrome c oxidase III, dTDP-glucose 4,6-dehydratase, γ-glutamyl transpeptidase and an abortive phage-resistance protein. At least some of these genes may be involved with colonization. A further six inserts had weak similarities to hypothetical proteins or to proteins with assigned functions from strain 11168. The remaining six clones had gene-fragment inserts with no database matches. Southern-blot analysis confirmed that strain-dependent variation existed for each of these DNA inserts. These results indicate that subtractive hybridization can successfully identify genes that are absent from the only C. jejuni strain for which the genome sequence is currently available.
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The benPK operon, proposed to play a role in transport, is part of a regulon for benzoate catabolism in Acinetobacter sp. strain ADP1
More LessBenM and CatM are distinct, but similar, LysR-type transcriptional regulators of the soil bacterium Acinetobacter sp. strain ADP1. Together, the two regulators control the expression of at least 14 genes involved in the degradation of aromatic compounds via the catechol branch of the β-ketoadipate pathway. In these studies, BenM and CatM were each purified to homogeneity to test the possibility that they regulate the expression of two additional genes, benP and benK, that are adjacent to benM on the chromosome. Each regulator bound to a DNA fragment containing the benP promoter region. Additional transcriptional studies suggested that benP and benK are co-transcribed as an operon, and a site of transcription initiation was identified. Alignment of this initiation site with those of several CatM- and BenM-regulated genes revealed common regulatory motifs. Mutants lacking both CatM and BenM failed to activate benP transcription. The ability of each protein to regulate gene expression was inferred from strains lacking either CatM or BenM that were still capable of increasing benP expression in response to cis,cis-muconate. This compound has previously been shown to induce all enzymes of the catechol branch of the β-ketoadipate pathway through a complex transcriptional circuit involving CatM and BenM. Thus, the regulated expression of the benPK operon in concert with other genes of the regulon is consistent with the model that BenP, a putative outer-membrane porin, and BenK, an inner-membrane permease, transport aromatic compounds in strain ADP1.
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Difference in substrate specificity divides the yeast alkali-metal-cation/H+ antiporters into two subfamilies
More LessYeast plasma membrane Na+/H+ antiporters (TC 2.A.36) share a high degree of similarity at the protein level. Expression of four antiporters (Saccharomyces cerevisiae Nha1p, Candida albicans Cnh1p, Zygosaccharomyces rouxii ZrSod2-22p and Schizosaccharomyces pombe sod2p) in a Sacch. cerevisiae mutant strain lacking both Na+-ATPase and Na+/H+ antiporter genes made it possible to study the transport properties and contribution to cell salt tolerance of all antiporters under the same conditions. The ZrSod2-22p of the osmotolerant yeast Z. rouxii has the highest transport capacity for lithium and sodium but, like the Schiz. pombe sod2p, it does not recognize K+ and Rb+ as substrates. The Sacch. cerevisiae Nha1p and C. albicans Cnh1p have a broad substrate specificity for at least four alkali metal cations (Na+, Li+, K+, Rb+), but their contribution to overall cell tolerance to high external concentration of toxic Na+ and Li+ cations seems to be lower compared to the antiporters of Schiz. pombe and especially Z. rouxii.
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Polymorphism in repeated 16S rRNA genes is a common property of type strains and environmental isolates of the genus Vibrio
More LessThe GenBank accession numbers for the sequences reported in this paper are AF388386 (Vp23), AF388387 (Vp16), AF388388 (F44), AF388389 (Vp27), AF388390 (F6), AF388391 (3d2), AF388392 (3d4), AF388393 (3d7) and AF388394 (3d8).
Analysis of the 16S rDNAs obtained from cultures of single colonies of either type collection strains or environmental strains of the genus Vibrio revealed the presence of polymorphism in every one of the strains examined. Polymorphism was detected by visualization of heteroduplexes produced after 16S rDNA PCR amplification, a procedure that allows for the screening of a large number of isolates. Amplified 16S rDNAs obtained from both Vibrio parahaemolyticus and an environmental strain were cloned. Their nucleotide sequences revealed differences of up to 2% among 16S rDNAs from the same strain. Polymorphic sites were concentrated in a recognized variable stem–loop of bacterial 16S rRNA that contained in some cases up to 83% of the total mismatches observed. Most of the substitutions present in the stem–loop region showed compensating base covariation. The accumulation of so many compensating changes in the stem–loop region implies that the divergence of the different versions of this stem–loop is relatively ancient. This divergence could be the result of either a selection process or a lateral transfer of independently evolved genes.
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