- Volume 154, Issue 10, 2008
Volume 154, Issue 10, 2008
- Mini-Review
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The chronicles of Porphyromonas gingivalis: the microbium, the human oral epithelium and their interplay
More LessThe microbiota of the human oral mucosa consists of a myriad of bacterial species that normally exist in commensal harmony with the host. Porphyromonas gingivalis, an aetiological agent in severe forms of periodontitis (a chronic inflammatory disease), is a prominent component of the oral microbiome and a successful colonizer of the oral epithelium. This Gram-negative anaerobe can also exist within the host epithelium without the existence of overt disease. Gingival epithelial cells, the outer lining of the gingival mucosa, which function as an important part of the innate immune system, are among the first host cells colonized by P. gingivalis. This review describes recent studies implicating the co-existence and intracellular adaptation of the organism in these target host cells. Specifically, recent findings on the putative mechanisms of persistence, intercellular dissemination and opportunism are highlighted. These new findings may also represent an original and valuable model for mechanistic characterization of other successful host-adapted, self-limiting, persistent intracellular bacteria in human epithelial tissues.
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- Cell And Developmental Biology
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The putrescine analogue 1,4-diamino-2-butanone affects polyamine synthesis, transport, ultrastructure and intracellular survival in Leishmania amazonensis
More LessPolyamines are important regulators of growth and differentiation in a variety of cells, including parasitic protozoa. Promastigotes of Leishmania species have high levels of putrescine and spermidine and their growth can be inhibited by polyamine biosynthesis antagonists. The putrescine analogue 1,4-diamino-2-butanone (DAB) is microbicidal against Tritrichomonas foetus and Trypanosoma cruzi, so we tested its effects on Leishmania amazonensis proliferation, viability, organization, putrescine transport and synthesis as well as in vitro infectivity. DAB impaired promastigote proliferation dose-dependently (IC50 144 μM) and the parasite putrescine concentration was reduced by nearly 50 %. This analogue markedly inhibited both ornithine decarboxylase activity and [H3]putrescine uptake by promastigotes. Pre-treatment with DAB for 24 h led to compensatory enhancement of putrescine uptake, indicating an adaptive mechanism in DAB-treated parasites. Remarkably, DAB caused mitochondrial damage, assessed by transmission electron microscopy, and 3 h treatment with 1 mM DAB enhanced lipid peroxidation, whereas incubation with 10 mM DAB or for 24 h resulted in decreased peroxidation levels in the parasites. This effect was probably due to the loss of mitochondrial function, demonstrated by the diminished reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), not observed in macrophages. Murine macrophages infected with L. amazonensis amastigotes treated with DAB had parasite loads significantly (P<0.05) lower than controls, presumably due to interference with putrescine uptake and/or synthesis. These results suggest that putrescine may be involved in leishmanial survival, possibly by maintaining the parasite's mitochondrial function. The use of analogues to interfere with polyamine/diamine synthesis and transport may shed light on its function in intracellular parasite survival and lead to identification of new targets for leishmaniasis chemotherapy.
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- Biochemistry And Molecular Biology
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Identification of TmcN as a pathway-specific positive regulator of tautomycetin biosynthesis in Streptomyces sp. CK4412
More LessTautomycetin (TMC) is a novel activated T-cell-specific immunosuppressive compound with a unique structure, containing an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. A 3 kb gene, tmcN, with a deduced product of 1029 amino acid residues, located on the 3′-terminus of an approximately 70 kb contiguous TMC biosynthetic gene cluster, was found to have amino acid sequence homology with bacterial regulatory proteins. In silico database comparisons revealed that TmcN belongs to the large ATP-binding regulators of the LuxR protein family. Gene disruption of tmcN from the Streptomyces sp. CK4412 chromosome resulted in significantly reduced antifungal activity against Aspergillus niger, as well as the absence of TMC. In addition, complementation by an integrative plasmid carrying tmcN restored TMC biosynthesis, strongly suggesting that TmcN is a positive regulator of TMC biosynthesis. Gene expression analysis by RT-PCR of the TMC biosynthetic genes revealed that a TmcN mutant strain exhibited reduced expression levels for most of the biosynthetic genes except for its own tmcN. It is thus suggested that TmcN is a pathway-specific positive regulator that activates transcription of the TMC biosynthetic pathway genes in Streptomyces sp. CK4412.
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Cyclic di-GMP: a second messenger required for long-term survival, but not for biofilm formation, in Mycobacterium smegmatis
More LessCyclic di-GMP (c-di-GMP) plays an important role in bacterial adaptation to enable survival in changing environments. It orchestrates various pathways involved in biofilm formation, changes in the cell surface, host colonization and virulence. In this article, we report the presence of c-di-GMP in Mycobacterium smegmatis, and its role in the long-term survival of the organism. M. smegmatis has a single bifunctional protein with both GGDEF and EAL domains, which show diguanylate cyclase (DGC) and phosphodiesterase (PDE)-A activity, respectively, in vitro. We named this protein MSDGC-1. Deletion of the gene encoding MSDGC-1 did not affect growth and biofilm formation in M. smegmatis, but long-term survival under conditions of nutritional starvation was affected. Most of the proteins that contain GGDEF and EAL domains have been demonstrated to have either DGC or PDE-A activity. To gain further insight into the regulation of the protein, we cloned the individual domains, and tested their respective activities. MSDGC-1, the full-length protein, is required for activity, as its GGDEF and EAL domains are inactive when separated.
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Phosphorylation-independent activation of the atypical response regulator NblR
Cyanobacteria respond to environmental stress conditions by adjusting their photosynthesis machinery. In Synechococcus sp. PCC 7942, phycobilisome degradation and other acclimation responses after nutrient or high-light stress require activation by the orphan response regulator NblR, a member of the OmpR/PhoB family. Although NblR contains a putative phosphorylatable residue (Asp57), it lacks other conserved residues required to chelate the Mg2+ necessary for aspartic acid phosphorylation or to transduce the phosphorylation signal. In close agreement with these features, NblR was not phosphorylated in vitro by the low-molecular-mass phosphate donor acetyl phosphate and mutation of Asp57 to Ala had no impact on previously characterized NblR functions in Synechococcus. On the other hand, in vitro and in vivo assays show that the default state of NblR is monomeric, suggesting that, despite input differences, NblR activation could involve the same general mechanism of activation by dimerization present in known members of the OmpR/PhoB family. Structural and functional data indicate that the receiver domain of NblR shares similarities with other phosphorylation-independent response regulators such as FrzS and HP1043. To acknowledge the peculiarities of these atypical ‘two-component’ regulators with phosphorylation-independent signal transduction mechanisms, we propose the term PIARR, standing for phosphorylation-independent activation of response regulator.
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Polar secretion of proteins via the Xcp type II secretion system in Pseudomonas aeruginosa
More LessThe subcellular localization of the major type II secretion system of Pseudomonas aeruginosa, the Xcp system, was studied microscopically using a biarsenical ligand that becomes fluorescent upon binding to a tetracysteine motif (Lumio tag), which was fused to several Xcp components. Fusion of the Lumio tag to the C termini of the XcpR and XcpS proteins did not affect the functionality of these proteins. Fluorescence microscopy showed that they were predominantly localized to the poles of P. aeruginosa cells, when produced at levels comparable to chromosomally encoded XcpR and XcpS. In most labelled cells, the proteins were found at one of the poles, although bipolar localization was also observed. When produced in the absence of other Xcp components, labelled XcpS was still found to locate at the poles, whereas XcpR was evenly distributed in the cell. These data suggest that XcpS, but not XcpR, contains information required for polar localization. The polar location of the Xcp machinery was further confirmed by the visualization of protease secretion with an intramolecularly quenched casein conjugate.
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Division protein interaction web: identification of a phylogenetically conserved common interactome between Streptococcus pneumoniae and Escherichia coli
The ability of each of the 11 Streptococcus pneumoniae division proteins to interact with itself and with each of the remaining proteins was studied in 66 combinations of protein pairs, using a bacterial two-hybrid system. Interactions (homo- or hetero-dimerizations) were detected between 37 protein pairs, whereas 29 protein pairs did not interact. In some cases, positive interactions of the S. pneumoniae proteins were confirmed by co-immunoprecipitation experiments in Escherichia coli. Comparison between the S. pneumoniae division protein interaction web and that of E. coli, the only micro-organisms for which the whole division interactome has been described systematically, was also performed. At least nine division proteins, ZapA, FtsZ, FtsA, FtsK, FtsQ/DivIB, FtsB/DivIC, FtsL, FtsI and FtsW, are believed to have a conserved function between these bacteria and thus we may say that a significant part of the interactions are conserved. Out of 45 protein pairs tested in both bacteria, 30 showed the same behaviour: 23 interacted while seven did not. In agreement with these results, cross-interactions between S. pneumoniae proteins and the corresponding E. coli orthologues were observed. Taken together, these results suggest a phylogenetically conserved minimal common interactome of the division proteins.
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Nitrate-responsive NarX-NarL represses arginine-mediated induction of the Pseudomonas aeruginosa arginine fermentation arcDABC operon
More LessDenitrification and arginine fermentation are major parts of the anaerobic metabolism of Pseudomonas aeruginosa, which is important for biofilm formation and infection. The two-component regulatory system NarX-NarL is part of the underlying network and is required for denitrifying growth. All target promoters identified so far are activated by NarL. In this study the effect of NarL on arginine fermentation was investigated using proteome, Northern blot and lacZ reporter gene analyses. NarL-dependent repression of the arcDABC operon was observed and the corresponding NarL-binding site in the arcD promoter region was functionally localized at −60 bp upstream of the transcriptional start site using site-directed promoter mutagenesis and reporter gene fusion experiments. The results clearly show that in the presence of nitrate NarL represses the arginine-dependent activation of the arcDABC operon mediated by ArgR. It does not influence the oxygen-tension-dependent activation via Anr. Thus, the anaerobic energy metabolism of P. aeruginosa is coordinated via NarX-NarL activity. In the presence of nitrate the highly efficient denitrification is preferred over the less attractive arginine fermentation.
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The activity of the glyoxylate cycle in peroxisomes of Candida albicans depends on a functional β-oxidation pathway: evidence for reduced metabolite transport across the peroxisomal membrane
The glyoxylate cycle, a metabolic pathway required for generating C4 units from C2 compounds, is an important factor in virulence, in both animal and plant pathogens. Here, we report the localization of the key enzymes of this cycle, isocitrate lyase (Icl1; EC 4.1.3.1) and malate synthase (Mls1; EC 2.3.3.9), in the human fungal pathogen Candida albicans. Immunocytochemistry in combination with subcellular fractionation showed that both Icl1 and Mls1 are localized to peroxisomes, independent of the carbon source used. Although Icl1 and Mls1 lack a consensus type I peroxisomal targeting signal (PTS1), their import into peroxisomes was dependent on the PTS1 receptor Pex5p, suggesting the presence of non-canonical targeting signals in both proteins. Peroxisomal compartmentalization of the glyoxylate cycle is not essential for proper functioning of this metabolic pathway because a pex5Δ/Δ strain, in which Icl1 and Mls1 were localized to the cytosol, grew equally as well as the wild-type strain on acetate and ethanol. Previously, we reported that a fox2Δ/Δ strain that is completely deficient in fatty acid β-oxidation, but has no peroxisomal protein import defect, displayed strongly reduced growth on non-fermentable carbon sources such as acetate and ethanol. Here, we show that growth of the fox2Δ/Δ strain on these carbon compounds can be restored when Icl1 and Mls1 are relocated to the cytosol by deleting the PEX5 gene. We hypothesize that the fox2Δ/Δ strain is disturbed in the transport of glyoxylate cycle products and/or acetyl-CoA across the peroxisomal membrane and discuss the possible relationship between such a transport defect and the presence of giant peroxisomes in the fox2Δ/Δ mutant.
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Effect of carbon source availability and growth phase on expression of Corynebacterium glutamicum genes involved in the tricarboxylic acid cycle and glyoxylate bypass
More LessThe effect of different carbon sources on the expression of tricarboxylic acid (TCA) cycle genes, along with glyoxylate bypass genes, in Corynebacterium glutamicum was determined. All TCA cycle genes were coordinately expressed in medium containing acetate. Growth in the presence of acetate gave rise to abundant expression of most TCA cycle genes, with the level of gltA transcript being the highest. However, when the cells entered the stationary phase triggered by acetate exhaustion, all genes were repressed, except sucCD and mdhB, which were slightly induced. Acetate withdrawal from the growth medium during the exponential phase also led to rapid repression of most TCA cycle genes and a corresponding twofold increase in the expression of sucCD, which were strongly induced by citrate and succinate. In addition, glucose depletion during the stationary phase led to a corresponding 8–20-fold induction of the sucCD, aceA and aceB genes. Addition of glucose to acetate medium resulted in about 10-fold induction of sucCD. The strong dependence of TCA cycle sucCD and glyoxylate bypass aceA and aceB expression on carbon source availability was confirmed and the regulatory system will be studied precisely.
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Analysis of the pSK1 replicon, a prototype from the staphylococcal multiresistance plasmid family
More LessMultidrug-resistant staphylococci often harbour plasmids that carry genes conferring resistance to several antimicrobial compounds. Many of these multiresistance plasmids appear to utilize a related theta-type replication system for which multiresistance plasmid pSK1 serves as a prototype. Essential pSK1 replication elements were identified by cloning segments of the replication region and testing the resulting plasmids for replication proficiency. An iterated region within rep and a DNA segment of up to 68 bp upstream of the rep promoter were both found to be essential for origin activity. The Rep protein was overexpressed as a 6×His-tagged C-terminal fusion protein and was shown to bind in vitro to four Rep boxes located within the rep coding region. Inactivation of a divergently oriented promoter upstream of rep, designated P rnaI , resulted in an elevated plasmid copy number. Comparative analyses suggest that the replication systems of many staphylococcal multiresistance plasmids share a similar genetic organization and utilize an antisense-RNA-mediated regulatory mechanism for copy number control.
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Aromatic degradative pathways in Acinetobacter baylyi underlie carbon catabolite repression
More LessCarbon catabolite repression is an important mechanism allowing efficient carbon source utilization. In the soil bacterium Acinetobacter baylyi, this mechanism has been shown to apply to the aromatic degradative pathways for the substrates protocatechuate, p-hydroxybenzoate and vanillate. In this investigation, transcriptional fusions with the gene for luciferase in the gene clusters for the degradation of benzyl esters, anthranilate, benzoate, hydroxycinnamates and dicarboxylates (are, ant, ben, hca and dca genes) were constructed and established in the chromosome of A. baylyi. The respective strains revealed the presence of strong carbon catabolite repression at the transcriptional level. In all cases, succinate and acetate in combination had the strongest repressing effect, and pyruvate (or lactate in case of the ben and hca genes) allowed the highest expression when these carbon sources were supplied together with the respective inducer. The pattern of repression for the different cosubstrates was similar for all operons investigated and was also observed in the absence of the respective inducing compounds, indicating a mechanism that is independent of the respective specific regulators. Repression by acetate and succinate varied between 88 % for the hca genes and 99 % for the pca genes.
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- Biodiversity And Evolution
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Diversity of IncP-9 plasmids of Pseudomonas
IncP-9 plasmids are important vehicles for degradation and resistance genes that contribute to the adaptability of Pseudomonas species in a variety of natural habitats. The three completely sequenced IncP-9 plasmids, pWW0, pDTG1 and NAH7, show extensive homology in replication, partitioning and transfer loci (an ∼25 kb region) and to a lesser extent in the remaining backbone segments. We used PCR, DNA sequencing, hybridization and phylogenetic analyses to investigate the genetic diversity of 30 IncP-9 plasmids as well as the possibility of recombination between plasmids belonging to this family. Phylogenetic analysis of rep and oriV sequences revealed nine plasmid subgroups with 7–35 % divergence between them. Only one phenotypic character was normally associated with each subgroup, except for the IncP-9β cluster, which included naphthalene- and toluene-degradation plasmids. The PCR and hybridization analysis using pWW0- and pDTG1-specific primers and probes targeting selected backbone loci showed that members of different IncP-9 subgroups have considerable similarity in their overall organization, supporting the existence of a conserved ancestral IncP-9 sequence. The results suggested that some IncP-9 plasmids are the product of recombination between plasmids of different IncP-9 subgroups but demonstrated clearly that insertion of degradative transposons has occurred on multiple occasions, indicating that association of this phenotype with these plasmids is not simply the result of divergent evolution from a single successful ancestral degradative plasmid.
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Development of an unambiguous and discriminatory multilocus sequence typing scheme for the Streptococcus zooepidemicus group
The zoonotic pathogen Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is commonly found harmlessly colonizing the equine nasopharynx. Occasionally, strains can invade host tissues or cross species barriers, and S. zooepidemicus is associated with numerous different diseases in a variety of hosts, including inflammatory airway disease and abortion in horses, pneumonia in dogs and meningitis in humans. A biovar of S. zooepidemicus, Streptococcus equi subsp. equi, is the causative agent of strangles, one of the most important infections of horses worldwide. We report here the development of the first multilocus sequence typing (MLST) scheme for S. zooepidemicus and its exploitation to define the population genetic structure of these related pathogens. A total of 130 unique sequence types were identified from 277 isolates of diverse geographical and temporal origin. Isolates of S. equi shared a recent evolutionary ancestor with isolates of S. zooepidemicus that were significantly associated with cases of uterine infection or abortion in horses (P<0.001). Isolates of S. zooepidemicus from three UK outbreaks of acute fatal haemorrhagic pneumonia in dogs during 1999, 2001 and 2008 were found to be related to isolates from three outbreaks of this disease in the USA during 2005, 1993 and 2006, respectively. Our data provide strong evidence that S. equi evolved from an ancestral S. zooepidemicus strain and that certain related strains of S. zooepidemicus have a greater propensity to infect particular hosts and tissues.
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- Environmental Microbiology
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Molecular analysis of the diversity of the sulfide : quinone reductase (sqr) gene in sediment environments
More LessOur newly designed primers were evaluated for the molecular analysis of specific groups of the sqr gene encoding sulfide : quinone reductase (SQR) in sediment environments. Based on the phylogenetic analysis, we classified the sqr sequences into six groups. PCR primers specific for each group were developed. We successfully amplified sqr-like gene sequences related to groups 1, 2 and 4 from diverse sediments including a marine sediment (SW), a tidal flat (TS), a river sediment (RS) and a lake sediment (FW). We recovered a total of 82 unique phylotypes (based on a 95 % amino acid sequence similarity cutoff) from 243 individual sqr-like gene sequences. Phylotype richness varied widely among the groups of sqr-like gene sequences (group 1>group 2>group 4) and sediments (SW>TS>RS>FW). Most of the sqr-like gene sequences were affiliated with the Proteobacteria clade and were distantly related to the reference sqr gene sequences from cultivated strains (less than ∼80 % amino acid sequence similarity). Unique sqr-like gene sequences were associated with individual sediment samples in groups 1 and 2. This molecular tool has also enabled us to detect sqr-like genes in a sulfur-oxidizing enrichment from marine sediments. Collectively, our results support the presence of previously unrecognized sqr gene-containing micro-organisms that play important roles in the global biogeochemical cycle of sulfur.
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Forces involved in bacterial adhesion to hydrophilic and hydrophobic surfaces
More LessUsing a parallel-plate flow chamber, the hydrodynamic shear forces to prevent bacterial adhesion (Fprev ) and to detach adhering bacteria (Fdet ) were evaluated for hydrophilic glass, hydrophobic, dimethyldichlorosilane (DDS)-coated glass and six different bacterial strains, in order to test the following three hypotheses. 1. A strong hydrodynamic shear force to prevent adhesion relates to a strong hydrodynamic shear force to detach an adhering organism. 2. A weak hydrodynamic shear force to detach adhering bacteria implies that more bacteria will be stimulated to detach by passing an air–liquid interface (an air bubble) through the flow chamber. 3. DLVO (Derjaguin, Landau, Verwey, Overbeek) interactions determine the characteristic hydrodynamic shear forces to prevent adhesion and to detach adhering micro-organisms as well as the detachment induced by a passing air–liquid interface. Fprev varied from 0.03 to 0.70 pN, while Fdet varied from 0.31 to over 19.64 pN, suggesting that after initial contact, strengthening of the bond occurs. Generally, it was more difficult to detach bacteria from DDS-coated glass than from hydrophilic glass, which was confirmed by air bubble detachment studies. Calculated attractive forces based on the DLVO theory (FDLVO ) towards the secondary interaction minimum were higher on glass than on DDS-coated glass. In general, all three hypotheses had to be rejected, showing that it is important to distinguish between forces acting parallel (hydrodynamic shear) and perpendicular (DLVO, air–liquid interface passages) to the substratum surface.
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- Genes And Genomes
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Characterization of Streptococcus gordonii prophage PH15: complete genome sequence and functional analysis of phage-encoded integrase and endolysin
More LessStreptococcus gordonii OMZ1039, isolated from supragingival dental plaque, was found to harbour a prophage, PH15, whose excision could be induced by mitomycin treatment. Phage PH15 belongs to the Siphoviridae. The complete genome sequence of PH15 was determined. The genome was 39 136 bp in size and contained 61 ORFs. The genome of PH15 was most similar in the structural module to the temperate bacteriophages MM1 and φNIH1.1 from Streptococcus pneumoniae and Streptococcus pyogenes, respectively. In strain OMZ1039, PH15 was found to reside as a prophage in the cysteinyl-tRNA gene. A plasmid, harbouring the attP site and the integrase gene downstream of a constitutive promoter, was capable of site-specific integration into the genomes of different oral streptococcal species. The phage endolysin was purified after expression in Escherichia coli and found to inhibit growth of all S. gordonii strains tested and several different streptococcal species, including the pathogens Streptococcus mutans, S. pyogenes and Streptococcus agalactiae.
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The cyanobacterial homologue of the RNA chaperone Hfq is essential for motility of Synechocystis sp. PCC 6803
The ssr3341 locus was previously suggested to encode an orthologue of the RNA chaperone Hfq in the cyanobacterium Synechocystis sp. strain PCC 6803. Insertional inactivation of this gene resulted in a mutant that was not naturally transformable and exhibited a non-phototactic phenotype compared with the wild-type. The loss of motility was complemented by reintroduction of the wild-type gene, correlated with the re-establishment of type IV pili on the cell surface. Microarray analyses revealed a small set of genes with drastically reduced transcript levels in the knockout mutant compared with the wild-type cells. Among the most strongly affected genes, slr1667, slr1668, slr2015, slr2016 and slr2018 stood out, as they belong to two operons that had previously been shown to be involved in motility, controlled by the cAMP receptor protein SYCRP1. This suggests a link between cAMP signalling, motility and possibly the involvement of RNA-based regulation. This is believed to be the first report demonstrating a functional role of an Hfq orthologue in cyanobacteria, establishing a new factor in the control of motility.
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Multidrug efflux pump overexpression in Staphylococcus aureus after single and multiple in vitro exposures to biocides and dyes
More LessBiocides and dyes are commonly employed in hospital and laboratory settings. Many of these agents are substrates for multiple-drug resistance (MDR)-conferring efflux pumps of both Gram-positive and Gram-negative organisms. Several such pumps have been identified in Staphylococcus aureus, and mutants overexpressing the NorA and MepA MDR pumps following exposure to fluoroquinolones have been identified. The effect of exposure to low concentrations of biocides and dyes on the expression of specific pump genes has not been evaluated. Using quantitative reverse-transcription PCR we found that exposure of clinical isolates to low concentrations of a variety of biocides and dyes in a single step, or to gradually increasing concentrations over several days, resulted in the appearance of mutants overexpressing mepA, mdeA, norA and norC, with mepA overexpression predominating. Overexpression was frequently associated with promoter-region or regulatory protein mutations. Mutants having significant increases in MICs of common pump substrates but no changes in expression of studied pump genes were also observed; in these cases changes in expression of as-yet-unidentified MDR pump genes may have occurred. Strains of S. aureus that exist in relatively protected environments and are repeatedly exposed to sublethal concentrations of biocides can develop efflux-related resistance to those agents, and acquisition of such strains poses a threat to patients treated with antimicrobial agents that are also substrates for those pumps, such as ciprofloxacin and moxifloxacin.
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Genetic prerequisites for additive or synergistic actions of 5-fluorocytosine and fluconazole in baker's yeast
More LessDuring applications of 5-fluorocytosine (5FC) and fluconazole (FLC), additive or synergistic action may even occur when primary resistance to 5FC is established. Here, we analysed conjoint drug action in Saccharomyces cerevisiae strains deficient in genes known to be essential for 5FC or FLC function. Despite clear primary resistance, residual 5FC activity and additive 5FC+FLC action in cells lacking cytosine permease (Fcy2p) or uracil phosphoribosyl transferase (Fur1p) were detected. In contrast, Δfcy1 mutants, lacking cytosine deaminase, became entirely resistant to 5FC, concomitantly losing 5FC+FLC additivity. Disruption of the orotate phosphoribosyltransferase gene (URA5) in the wild-type led to low-level 5FC tolerance, while an alternative orotate phosphoribosyltransferase, encoded by URA10, contributed to 5FC toxicity only in the Δura5 background. Remarkably, combination of Δura5 and Δfur1 resulted in complete 5FC resistance. Thus, yeast orotate phosphoribosyltransferases are involved in 5FC metabolism. Similarly, disruption of the ergosterol Δ5,6-desaturase-encoding gene ERG3 resulted only in partial resistance to FLC, and concomitantly a synergistic effect with 5FC became evident. Full resistance to FLC occurred in Δerg3 Δerg11 double mutants and, simultaneously, synergism or even an additive effect with FLC and 5FC was no longer discernible. Since the majority of spontaneously occurring resistant yeast clones displayed residual sensitivity to either 5FC or FLC and those strains responded to combined drug treatment in a predictable manner, careful resistance profiling based on the findings reported here may help to address yeast infections by combined application of antimycotic compounds.
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Identification and use of the putative Bacteroides ovatus xylanase promoter for the inducible production of recombinant human proteins
The use of genetically modified bacteria to deliver biologically active molecules directly to the gut has become an increasingly attractive area of investigation. The challenge of regulation of production of the therapeutic molecule and colonization of the bowel led us to investigate Bacteroides ovatus for the production of these molecules, due to its ability to colonize the colon and xylan utilization properties. Here we have identified the putative xylanase promoter. The 5′ region of the corresponding mRNA was determined by 5′RACE analysis and the transcription initiation site was identified 216 bp upstream of the ATG start codon. The putative xylanase promoter was regulated by xylan in a dose- and time-dependent manner, and repressed by glucose. This promoter was subsequently used to direct the controlled expression of a gene encoding the human intestinal trefoil factor (TFF-3) after integration as a single copy into the chromosome of B. ovatus. The resulting strain produced biologically active TFF-3 in the presence of xylan. These findings identify the B. ovatus xylanase operon promoter and show that it can be utilized to direct xylan-inducible expression of heterologous eukaryotic genes in B. ovatus.
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Detection of small RNAs in Pseudomonas aeruginosa by RNomics and structure-based bioinformatic tools
Inactivation of the Pseudomonas aeruginosa (PAO1) hfq gene, encoding the Sm-like Hfq protein, resulted in pleiotropic effects that included an attenuated virulence. As regulation by Hfq often involves the action of small regulatory RNAs (sRNAs), we have used a shotgun cloning approach (RNomics) and bioinformatic tools to identify sRNAs in strain PAO1. For cDNA library construction, total RNA was extracted from PAO1 cultures either grown to stationary phase or exposed to human serum. The cDNA libraries were generated from small-sized RNAs of PAO1 after co-immunoprecipitation with Hfq. Of 400 sequenced cDNA clones, 11 mapped to intergenic regions. Band-shift assays and Northern blot analyses performed with two selected sRNAs confirmed that Hfq binds to and affects the steady-state levels of these RNAs. A proteome study performed upon overproduction of one sRNA, PhrS, implicated it in riboregulation. PhrS contains an ORF, and evidence for its translation is presented. In addition, based on surveys with structure-based bioinformatic tools, we provide an electronic compilation of putative sRNA and non-coding RNA genes of PAO1 based on their evolutionarily conserved structure.
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- Pathogens And Pathogenicity
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The role of the RgpA–Kgp proteinase–adhesin complexes in the adherence of Porphyromonas gingivalis to fibroblasts
Porphyromonas gingivalis strains W50 and ATCC 33277 were shown to bind to cultured human fibroblast (MRC-5) cells using flow cytometry. As the concentration of P. gingivalis strain W50 cells was increased relative to the concentration of MRC-5 cells, the number of W50 cells bound per MRC-5 cell increased, as did the percentage of MRC-5 cells with bacteria bound. However, this relationship was only seen for P. gingivalis strain ATCC 33277 at low cell concentrations: at high bacterial cell concentrations strain ATCC 33277 auto-aggregated and binding to the MRC-5 cells decreased. Strain W50 was therefore chosen to study the role of the surface proteinase–adhesin complexes (RgpA–Kgp complexes) in binding to MRC-5 cells. P. gingivalis W50 cells treated with an inhibitor of the RgpA–Kgp complexes exhibited reduced binding to MRC-5 cells. The purified active and proteinase-inactive RgpA–Kgp complexes competitively inhibited binding of W50 to MRC-5 cells, and isogenic mutants of W50 lacking RgpA/B and Kgp displayed reduced binding. P. gingivalis W50 mutant cells lacking Kgp exhibited the lowest binding to MRC-5 cells, suggesting an important role for this proteinase and its associated adhesins in binding to fibroblasts.
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LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest
The success of Mycobacterium tuberculosis depends on its ability to survive within host macrophages. Here, M. tuberculosis avoids the acidic, hydrolytically competent environment of the phagolysosome by arresting phagosome maturation. Having shown previously that a M. tuberculosis mutant deficient in lipoprotein signal peptidase (LspA) is strongly attenuated in vivo in a mouse model of infection, we now studied putative mechanisms involved in attenuation of the lspA : : aph mutant at a cellular level. In this work we investigated the ability of the mutant to interfere with two host defence mechanisms, i.e. Toll-like receptor (TLR)2-dependent immune response and phagosome maturation. While mycobacterial lipoproteins have been reported to trigger a TLR2 signalling pathway critical for innate immune responses, we found that growth control of the lspA : : aph mutant was independent of TLR2. In addition, the lspA : : aph mutant arrested phagosome maturation to an extent similar to that of the wild-type, as measured by lysosomal-associated membrane protein 1 (LAMP1) co-localization and intraphagosomal pH. These observations demonstrate severe attenuation even in the presence of arrested phagosome maturation, and point to a role for the early phagosome in growth restriction of the M. tuberculosis lspA mutant.
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Interaction of Mycoplasma genitalium with host cells: evidence for nuclear localization
Mycoplasma genitalium (Mg) is a mollicute that causes a range of human urogenital infections. A hallmark of these bacteria is their ability to establish chronic infections that can persist despite completion of appropriate antibiotic therapies and intact and functional immune systems. Intimate adherence and surface colonization of mycoplasmas to host cells are important pathogenic features. However, their facultative intracellular nature is poorly understood, partly due to difficulties in developing and standardizing cellular interaction model systems. Here, we characterize growth and invasion properties of two Mg strains (G37 and 1019V). Mg G37 is a high-passage laboratory strain, while Mg 1019V is a low-passage isolate recovered from the cervix. The two strains diverge partially in gene sequences for adherence-related proteins and exhibit subtle variations in their axenic growth. However, with both strains and consistent with our previous studies, a subset of adherent Mg organisms invade host cells and exhibit perinuclear targeting. Remarkably, intranuclear localization of Mg proteins is observed, which occurred as early as 30 min after infection. Mg strains deficient in adherence were markedly reduced in their ability to invade and associate with perinuclear and nuclear sites.
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Role of Mycoplasma genitalium MG218 and MG317 cytoskeletal proteins in terminal organelle organization, gliding motility and cytadherence
More LessThe terminal organelle is a differentiated structure that plays a key role in mycoplasma cytadherence and locomotion. For this reason, the analysis of Mycoplasma genitalium mutants displaying anomalous terminal organelles could improve our knowledge regarding the structural elements required for proper locomotion. In this study, we isolated several M. genitalium mutants having transposon insertions within the mg218 or mg317 genes, which encode the orthologues of Mycoplasma pneumoniae HMW2 and HMW3 cytoskeletal proteins, respectively. As expected, mg218− and mg317− mutants exhibit a reduced gliding motility, although their ability to attach to solid surfaces was not completely abolished. Interestingly, most of the mg218− mutants expressed N-terminal MG218 derivatives and showed the presence of short terminal organelles retaining many of the functions displayed by this structure in the wild-type strain, suggesting that the N-terminal region of this protein is an essential element in the architecture of the terminal organelle. Separately, the analysis of mg317− mutants indicates that MG317 protein is involved in the formation of the terminal button and contributes to anchoring the electron-dense core to the cell membrane. The results presented here clearly show that MG218 and MG317 proteins are implicated in the maintenance of gliding motility and cytadherence in M. genitalium.
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Identification and phenotypic characterization of a second collagen adhesin, Scm, and genome-based identification and analysis of 13 other predicted MSCRAMMs, including four distinct pilus loci, in Enterococcus faecium
Attention has recently been drawn to Enterococcus faecium because of an increasing number of nosocomial infections caused by this species and its resistance to multiple antibacterial agents. However, relatively little is known about the pathogenic determinants of this organism. We have previously identified a cell-wall-anchored collagen adhesin, Acm, produced by some isolates of E. faecium, and a secreted antigen, SagA, exhibiting broad-spectrum binding to extracellular matrix proteins. Here, we analysed the draft genome of strain TX0016 for potential microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Genome-based bioinformatics identified 22 predicted cell-wall-anchored E. faecium surface proteins (Fms), of which 15 (including Acm) had characteristics typical of MSCRAMMs, including predicted folding into a modular architecture with multiple immunoglobulin-like domains. Functional characterization of one [Fms10; redesignated second collagen adhesin of E. faecium (Scm)] revealed that recombinant Scm65 (A- and B-domains) and Scm36 (A-domain) bound to collagen type V efficiently in a concentration-dependent manner, bound considerably less to collagen type I and fibrinogen, and differed from Acm in their binding specificities to collagen types IV and V. Results from far-UV circular dichroism measurements of recombinant Scm36 and of Acm37 indicated that these proteins were rich in β-sheets, supporting our folding predictions. Whole-cell ELISA and FACS analyses unambiguously demonstrated surface expression of Scm in most E. faecium isolates. Strikingly, 11 of the 15 predicted MSCRAMMs clustered in four loci, each with a class C sortase gene; nine of these showed similarity to Enterococcus faecalis Ebp pilus subunits and also contained motifs essential for pilus assembly. Antibodies against one of the predicted major pilus proteins, Fms9 (redesignated EbpCfm), detected a ‘ladder’ pattern of high-molecular-mass protein bands in a Western blot analysis of cell surface extracts from E. faecium, suggesting that EbpCfm is polymerized into a pilus structure. Further analysis of the transcripts of the corresponding gene cluster indicated that fms1 (ebpAfm ), fms5 (ebpBfm ) and ebpCfm are co-transcribed, a result consistent with those for pilus-encoding gene clusters of other Gram-positive bacteria. All 15 genes occurred frequently in 30 clinically derived diverse E. faecium isolates tested. The common occurrence of MSCRAMM- and pilus-encoding genes and the presence of a second collagen-binding protein may have important implications for our understanding of this emerging pathogen.
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Expression of two distinct types of pili by a hospital-acquired Enterococcus faecium isolate
Surface filamentous structures designated pili, and implicated in virulence, have been found on the surfaces of several Gram-positive pathogens. This work describes the conditional expression of two phenotypically distinct pilus-like structures, designated PilA and PilB, on the surface of a hospital-adapted Enterococcus faecium bloodstream isolate. E. faecium is an emerging Gram-positive opportunistic pathogen that can cause severe disease, particularly in immunocompromised patients. Expression of PilA- and PilB-type pili was analysed during different phases of growth in broth culture. During growth, PilA and PilB pilin subunits were expressed around the cross-wall in early-exponential-phase cells. Polymerization and migration of short PilB-type pili towards the poles occurred in cells from the exponential phase and long polymerized pili were expressed at the poles of cells grown to stationary phase. In contrast, PilA-type pili were not expressed in broth culture, but only when cells were grown on solid media. Furthermore, surface expression of the PilA- and PilB-type pili was regulated in a temperature-dependent manner, as polymerization of two distinct types of pili at the surface only occurred when cells were grown at 37 °C; no pili were observed on cells grown at 21 °C. Hospital-aquired E. faecium isolates were specifically enriched in pilin gene clusters, suggesting that conditional expression of pili may contribute to E. faecium pathogenesis.
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- Physiology
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The PA4204 gene encodes a periplasmic gluconolactonase (PpgL) which is important for fitness of Pseudomonas aeruginosa
In Pseudomonas aeruginosa, the PA4204 gene encodes a protein with a signal peptide and a COG2706 domain of the type present in 3-carboxy-cis,cis-muconate lactonizing enzymes. A molecular model based on the structure of the Escherichia coli YbhE phosphogluconate lactonizing enzyme shows that the enzyme has a beta-propeller (‘doughnut’) structure and a central active site comprising one histidine, one glutamic acid and two arginines. Inactivation of the P. aeruginosa PA4204 gene had profound phenotypic effects, resulting in slowly growing small colonies which frequently gave rise to larger colonies. The small colonies did not produce pyocyanin, produced reduced amounts of N-acylhomoserine lactones, and had extremely low levels of 2-alkyl-4-quinolones (AQs), while the larger colonies produced pyocyanin and higher amounts of AQs, including the pseudomonas quinolone signal (PQS), compared with the wild-type strain. Mutagenesis of His 182 in PA4204 resulted in the inability of this protein to restore pyocyanin production in the PA4204 isogenic mutant, suggesting that this enzyme may share an active site with other lactonizing enzymes. The protein with signal peptide was expressed as a His fusion in E. coli and purified. Two forms were observed, suggesting that the protein is translocated. The purified enzyme cleaved (S)-5-oxo-2-tetrahydrofurancarboxylic acid and d-glucono-δ-lactone, demonstrating lactonase activity. Decreased expression of the cytoplasmic phosphogluconolactonase gene (pgl) was observed in the small-colony mutant, and the mutant could not grow in the presence of mannitol or gluconate, suggesting functions in the detoxification of a gluconolactone and in sugar metabolism.
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Vancomycin heteroresistance and biofilm formation in Staphylococcus epidermidis from food
More LessStaphylococcus epidermidis CNBL 7032 is a heteroresistant strain, with subpopulations resistant to vancomycin concentrations up to 32 mg l −1, which was isolated from cured pork meat. The mechanisms of glycopeptide resistance in this strain were investigated in this study. S. epidermidis CNBL 7032 does not harbour enterococcal transmissible vancomycin-resistance genes. Transmission electron microscopy revealed that resistant subpopulations have a thicker cell wall, and that the increase in cell wall thickness is proportional to vancomycin concentration in the growth medium. Scanning electron microscopy showed that S. epidermidis CNBL 7032 forms a biofilm-like structure when grown in the presence of vancomycin. This food isolate harbours the gene atlE, coding for an autolysin with an adhesive function, which is involved in the first phase of biofilm formation. This study has demonstrated an interaction between atlE expression, biofilm formation and glycopeptide antibiotic resistance; transcription analysis demonstrated that the expression of atlE increased proportionally with the vancomycin concentration in the culture. Insertional inactivation of atlE confirmed the role of the AtlE autolysin in biofilm and vancomycin resistance.
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- Plant-Microbe Interactions
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The hrp genes of Pseudomonas cichorii are essential for pathogenicity on eggplant but not on lettuce
Pseudomonas cichorii causes necrotic lesions in eggplant and rot in lettuce. Through transposon insertion into P. cichorii strain SPC9018 we produced two mutants, 4-57 and 2-99, that lost virulence on eggplant but not lettuce. Analyses showed that a transposon was inserted into the hrpG gene in 4-57 and the hrcT gene in 2-99. Nucleotide sequences of the hrp genes of SPC9018 are homologous to those of Pseudomonas viridiflava BS group strains. The pathogenicity of 4-57 on eggplant was restored by transformation with an hrpF operon, originating from either SPC9018 or the BS group member P. viridiflava strain 9504 (Pv9504). These data suggested the involvement of hrp genes in the pathogenicity of SPC9018 on eggplant, and functional conservation of hrpF operons between SPC9018 and Pv9504. Both the hrpS mutant and the hrpL mutant were unable to cause necrotic lesions on eggplant leaves but retained their pathogenicity against lettuce. These results suggest that the pathogenicity of P. cichorii is hrp-dependent in eggplant, but not in lettuce.
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Characterizing the replication and stability regions of Spiroplasma citri plasmids identifies a novel replication protein and expands the genetic toolbox for plant-pathogenic spiroplasmas
More LessSpiroplasma citri strain GII3 contains seven plasmids, pSciA and pSci1–6, that share extensive regions of sequence homology and display a mosaic gene organization. Plasmid pSci2 comprises 12 coding sequences (CDS), three of which encode polypeptides homologous to proteins Soj/ParA, involved in chromosome partitioning, and TrsE and Mob/TraG, implicated in the type IV secretion pathway. One CDS encodes the adhesin-like protein ScARP3d whereas the other eight encode polypeptides with no homology to known proteins. The pSci2 CDS pE and soj have counterparts in all seven plasmids. Through successive deletions, various pSci2 derivatives were constructed and assessed for their ability to replicate by transformation of S. citri 44, a strain which has no plasmid. The smallest functional replicon was found to contain a single CDS (pE) and its flanking intergenic regions. Shuttle (S. citri/Escherichia coli) plasmids, in which CDS pE was disrupted, failed to replicate in S. citri, suggesting that PE is the replication protein of the S. citri plasmids. Successive propagations of pSci2-derived transformed spiroplasmas, in the absence of selection pressure, revealed that only pSci2 derivatives having an intact soj gene were stably maintained, indicating that the soj-encoded polypeptide is most likely involved in plasmid partitioning. Upon transformation, pSci2 derivatives, including shuttle (S. citri/E. coli) plasmids, were shown to replicate in all S. citri strains tested regardless of whether the strain possesses endogenous plasmids, such as strain GII3, or not, such as strain R8A2. In addition, the pSci replicons were introduced efficiently into the plant-pathogenic spiroplasmas Spiroplasma kunkelii and Spiroplasma phoeniceum, the transformation of which had never, to our knowledge, been described before. These studies show that, besides their implications for the biology of S. citri, the pSci plasmids hold considerable promise as vectors of general use for genetic studies of plant-pathogenic spiroplasmas. As an example, a HA-tagged S. citri protein was expressed in S. kunkelii. Detection of pE-hybridizing sequences in various group I spiroplasma species indicated that pE replicating plasmids were not restricted to the three plant-pathogenic spiroplasmas.
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- Theoretical Microbiology
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A mathematical model for growth and osmoregulation in halophilic bacteria
More LessMany molecular details of the ecophysiology of halophilic bacteria that use compatible solutes to maintain osmotic equilibrium have been examined. We ask whether the details are consistent and complete enough to predict growth and osmoregulation in these bacteria by integrating this information in a mathematical model. Parameterized for the halophilic organism Halomonas elongata, the model predicts the substrate and salt dependence of growth, the uptake of potassium and ectoine and the synthesis of ectoine. It is shown that salt (NaCl) dependence of growth can be modelled by substrate inhibition kinetics. Osmoregulation is known to involve accumulation of both ectoine and potassium glutamate in H. elongata. Using published and newly determined parameters, osmoregulatory models using either direct turgor or two-step (turgor and potassium) signalling are compared. The results are consistent with a role for potassium as a second messenger for hyperosmotic stress. Simulations of osmotic upshifts show a transient overregulation of the intracellular solute levels, as has been previously observed in experiments. A possible adaptive value of this overregulation as ‘pre-emptive’ behaviour in an environment with frequent dry periods leading to steadily increasing osmolarity is proposed. As a result of growth parameter estimation, a maximum P : O value of 2 for H. elongata can be inferred. In conclusion, the model developed here reproduces essential aspects of growth and osmoregulation in halophilic bacteria with a minimal set of assumptions.
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