- Volume 153, Issue 12, 2007
Volume 153, Issue 12, 2007
- Mini-Review
-
-
-
Making sense of quorum sensing in lactobacilli: a special focus on Lactobacillus plantarum WCFS1
More LessIn silico identification criteria were defined to predict if genes encoding histidine protein kinases (HPKs) and response regulators (RRs) could be part of peptide-based quorum sensing (QS) two-component regulatory systems (QS-TCSs) in Firmicutes. These criteria were used to screen HPKs and RRs annotated on the completed genome sequences of Lactobacillus species, and several (putative) QS-TCSs were identified in this way. The five peptide-based QS-TCSs that were predicted on the Lactobacillus plantarum WCFS1 genome were further analysed to test their (QS) functionality. Four of these systems contained an upstream gene encoding a putative autoinducing peptide (AIP), of which two were preceded by a double-glycine-type leader peptide. One of these was identical to the plnABCD regulatory system of L. plantarum C11 and was shown to regulate plantaricin production in L. plantarum WCFS1. The third TCS was designated lamBDCA for Lactobacillus agr-like module, where the lamD gene was shown to encode a cyclic thiolactone peptide. The fourth TCS was paralogous to the lam system and contained a putative AIP-encoding gene but lacked the lamB gene. Finally, a genetically separated orphan HPK and RR that showed clear peptide-based QS characteristics could form a fifth peptide-based QS-TCS. The predicted presence of multiple (peptide-based) QS-TCSs in some lactobacilli and in particular in L. plantarum might be a reflection of the ability of these species to persist in a diverse range of ecological niches.
-
-
-
-
The role of protein secretion systems in the virulence of the intracellular pathogen Legionella pneumophila
More LessLegionella pneumophila is a Gram-negative facultative intracellular pathogen, which multiplies in protozoa in its natural environment and can cause Legionnaires' disease in man, following infection of alveolar macrophages. In each of the different stages of infection of host cells, virulence proteins need to be delivered to their specific place of action and therefore must cross two barriers: the inner and the outer membrane. To date, several specialized secretion machineries for transport of proteins across the inner and outer membrane have been identified in L. pneumophila. Most of these secretion pathways have been shown to affect the virulence of this pathogen. An overview will be given of all the secretion pathways and the proteins transported by these secretion systems identified so far, with special attention paid to those that play a role in the pathogenicity of L. pneumophila.
-
- Sgm Special Lecture
-
-
-
Quorum sensing, communication and cross-kingdom signalling in the bacterial world
More LessAlthough unicellular, bacteria are highly interactive and employ a range of cell-to-cell communication or ‘quorum sensing (QS)’ systems for promoting collective behaviour within a population. QS is generally considered to facilitate gene expression only when the population has reached a sufficient cell density and depends on the synthesis of small molecules that diffuse in and out of bacterial cells. As the bacterial population density increases, so does the synthesis of QS signal molecules and consequently, their concentration in the external environment increases. Once a critical threshold concentration is reached, a target sensor kinase or response regulator is activated, so facilitating the expression of QS-dependent target genes. Several chemically distinct families of QS signal molecules have been described, of which the N-acylhomoserine lactone (AHL) family in Gram-negative bacteria have been the most intensively investigated. QS contributes to environmental adaptation by facilitating the elaboration of virulence determinants in pathogenic species and plant biocontrol characteristics in beneficial species as well as directing biofilm formation and colony escape. QS also crosses the prokaryotic–eukaryotic boundary in that QS signal molecules influence the behaviour of eukaryotic organisms in both the plant and mammalian worlds such that QS signal molecules may directly facilitate bacterial survival by promoting an advantageous lifestyle within a given environmental niche.
-
-
- Cell And Developmental Biology
-
-
-
The voltage-gated Na+ channel NaVBP co-localizes with methyl-accepting chemotaxis protein at cell poles of alkaliphilic Bacillus pseudofirmus OF4
NaVBP, found in alkaliphilic Bacillus pseudofirmus OF4, is a member of the bacterial voltage-gated Na+ channel superfamily. The alkaliphile requires NaVBP for normal chemotaxis responses and for optimal pH homeostasis during a shift to alkaline conditions at suboptimally low Na+ concentrations. We hypothesized that interaction of NaVBP with one or more other proteins in vivo, specifically methyl-accepting chemotaxis proteins (MCPs), is involved in activation of the channel under the pH conditions that exist in the extremophile and could underpin its role in chemotaxis; MCPs transduce chemotactic signals and generally localize to cell poles of rod-shaped cells. Here, immunofluorescence microscopy and fluorescent protein fusion studies showed that an alkaliphile protein (designated McpX) that cross-reacts with antibodies raised against Bacillus subtilis McpB co-localizes with NaVBP at the cell poles of B. pseudofirmus OF4. In a mutant in which NaVBP-encoding ncbA is deleted, the content of McpX was close to the wild-type level but McpX was significantly delocalized. A mutant of B. pseudofirmus OF4 was constructed in which cheAW expression was disrupted to assess whether this mutation impaired polar localization of McpX, as expected from studies in Escherichia coli and Salmonella, and, if so, whether NaVBP would be similarly affected. Polar localization of both McpX and NaVBP was decreased in the cheAW mutant. The results suggest interactions between McpX and NaVBP that affect their co-localization. The inverse chemotaxis phenotype of ncbA mutants may result in part from MCP delocalization.
-
-
- Biochemistry And Molecular Biology
-
-
-
Role of the methylcitrate cycle in propionate metabolism and detoxification in Mycobacterium smegmatis
More LessCatabolism of odd-chain-length fatty acids yields acetyl-CoA and propionyl-CoA. A common pathway of propionyl-CoA metabolism in micro-organisms is the methylcitrate cycle, which includes the dedicated enzymes methylcitrate synthase (MCS), methylcitrate dehydratase (MCD) and methylisocitrate lyase (MCL). The methylcitrate cycle is essential for propionate metabolism in Mycobacterium tuberculosis. Unusually, M. tuberculosis lacks an MCL orthologue and this activity is provided instead by two isoforms of the glyoxylate cycle enzyme isocitrate lyase (ICL1 and ICL2). These bifunctional (ICL/MCL) enzymes are jointly required for propionate metabolism and for growth and survival in mice. In contrast, the non-pathogenic species Mycobacterium smegmatis encodes a canonical MCL enzyme in addition to ICL1 and ICL2. The M. smegmatis gene encoding MCL (prpB) is clustered with genes encoding MCS (prpC) and MCD (prpD). Here we show that deletion of the M. smegmatis prpDBC locus reduced but did not eliminate MCL activity in cell-free extracts. The residual MCL activity was abolished by deletion of icl1 and icl2 in the ΔprpDBC background, suggesting that these genes encode bifunctional ICL/MCL enzymes. A ΔprpB Δicl1 Δicl2 mutant was unable to grow on propionate or mixtures of propionate and glucose. We hypothesize that incomplete propionyl-CoA metabolism might cause toxic metabolites to accumulate. Consistent with this idea, deletion of prpC and prpD in the ΔprpB Δicl1 Δicl2 background paradoxically restored growth on propionate-containing media. These observations suggest that the marked attenuation of ICL1/ICL2-deficient M. tuberculosis in mice could be due to the accumulation of toxic propionyl-CoA metabolites, rather than inability to utilize fatty acids per se.
-
-
-
-
Role of the C-terminal region of dextransucrase from Leuconostoc mesenteroides IBT-PQ in cell anchoring
More LessdsrP, a gene that encodes a cell-associated dextransucrase produced by Leuconostoc mesenteroides IBT-PQ, was isolated, sequenced and expressed in Escherichia coli. From sequence analysis, seven repeat units in the N-terminal region were found, as well as five cell wall binding repeats in the C-terminal region. A model of the C-terminal domain of dextransucrase was built based on the solenoid structure of the cell wall binding domain already described in LytA. By experiments involving direct interactions of the enzyme with L. mesenteroides cells, as well as among the cells and the single C-terminal domain expressed in E. coli, evidence was obtained concerning the anchoring function of this region in cell-associated dextransucrase, a function which may be independent of its capacity to bind dextran.
-
-
-
Phylogenetic and biochemical characterization of a novel cluster of intracellular fungal α-amylase enzymes
More LessCurrently known fungal α-amylases are well-characterized extracellular enzymes that are classified into glycoside hydrolase subfamily GH13_1. This study describes the identification, and phylogenetic and biochemical analysis of novel intracellular fungal α-amylases. The phylogenetic analysis shows that they cluster in the recently identified subfamily GH13_5 and display very low similarity to fungal α-amylases of family GH13_1. Homologues of these intracellular enzymes are present in the genome sequences of all filamentous fungi studied, including ascomycetes and basidiomycetes. One of the enzymes belonging to this new group, Amy1p from Histoplasma capsulatum, has recently been functionally linked to the formation of cell wall α-glucan. To study the biochemical characteristics of this novel cluster of α-amylases, we overexpressed and purified a homologue from Aspergillus niger, AmyD, and studied its activity product profile with starch and related substrates. AmyD has a relatively low hydrolysing activity on starch (2.2 U mg−1), producing mainly maltotriose. A possible function of these enzymes in relation to cell wall α-glucan synthesis is discussed.
-
-
-
A novel role for the yeast protein kinase Dbf2p in vacuolar H+-ATPase function and sorbic acid stress tolerance
More LessIn Saccharomyces cerevisiae, the serine-threonine protein kinase activity of Dbf2p is required for tolerance to the weak organic acid sorbic acid. Here we show that Dbf2p is required for normal phosphorylation of the vacuolar H+-ATPase (V-ATPase) A and B subunits Vma1p and Vma2p. Loss of V-ATPase activity due to bafilomycin treatment or deletion of either VMA1 or VMA2 resulted in sorbic acid hypersensitivity and impaired vacuolar acidification, phenotypes also observed in both a kinase-inactive dbf2 mutant and cells completely lacking DBF2 (dbf2Δ). Crucially, VMA2 is a multicopy suppressor of both the sorbic acid-sensitive phenotype and the impaired vacuolar-acidification defect of dbf2Δ cells, confirming a functional interaction between Dbf2p and Vma2p. The yeast V-ATPase is therefore involved in mediating sorbic acid stress tolerance, and we have shown a novel and unexpected role for the cell cycle-regulated protein kinase Dbf2p in promoting V-ATPase function.
-
-
-
Characterization of the mycobacterial chromosome segregation protein ParB and identification of its target in Mycobacterium smegmatis
Bacterial chromosomes (though not Escherichia coli and some other γ-proteobacterial chromosomes) contain parS sequences and parAB genes encoding partitioning proteins, i.e. ParA (ATPase) and ParB (DNA-binding proteins) that are components of the segregation machinery. Here, mycobacterial parABS elements were characterized for the first time. parAB genes are not essential in Mycobacterium smegmatis; however, elimination or overexpression of ParB protein causes growth inhibition. Deletion of parB also leads to a rather severe chromosome segregation defect: up to 10 % of the cells were anucleate. Mycobacterial ParB protein uses three oriC-proximal parS sequences as targets to organize the origin region into a compact nucleoprotein complex. Formation of such a complex involves ParB–ParB interactions and is assisted by ParA protein.
-
-
-
Consequences of a sortase A mutation in Streptococcus gordonii
Sortase A (SrtA) is required for cell-wall anchoring of LPXTG-containing Gram-positive surface proteins. It was hypothesized, therefore, that disruption of the srtA gene would alter surface anchoring and functions of target LPXTG motif-bearing SspA and SspB proteins of Streptococcus gordonii. Mutant strains in srtA (V288srtA −, DL1srtA− ) were constructed in S. gordonii V288 (wtV288) and DL1 (wtDL1). When compared to wtV288, the V288srtA− mutant showed decreased biofilm formation on polystyrene, and reduced binding to immobilized purified salivary agglutinin (BIAcore analysis). The wtV288 and V288srtA− strains were similar in ultrastructure, but immunogold-labelled SspA/SspB surface expression was reduced on the V288srtA− mutant. DL1srtA− was also complemented to obtain DL1srtA+ . From the wild-type strains (wtV288, wtDL1), srtA− mutants (V288srtA− , DL1srtA− ), and the complemented mutant (DL1srtA+ ), cytoplasmic, cell-wall and released extracellular protein fractions were isolated. Each fraction was analysed by SDS-PAGE and immunoblotting with anti-P1. Spent medium from srtA− mutant cells contained over-represented proteins, including SspA/SspB (P1 antigen). Mutants showed less P1 on the cell surface than wild-types, as estimated using whole-cell ELISA, and no P1 appeared in the cytoplasmic fractions. Expression of several adhesin genes (sspA/B, cshA/B, fbpA) was generally upregulated in the mutants (V288srtA− , DL1srtA− ), but restored to wild-type levels in DL1srtA+ . These data therefore imply that in addition to its role in processing LPXTG-containing adhesins, sortase A has the novel function of contributing to transcriptional regulation of adhesin gene expression.
-
-
-
Identification of multiple integration sites for Stx-phage Φ24B in the Escherichia coli genome, description of a novel integrase and evidence for a functional anti-repressor
The key virulence factor in Shiga-toxigenic Escherichia coli is the expression of Shiga toxin (Stx), which is conferred by Stx-encoding temperate lambdoid phages (Stx-phages). It had been assumed that Stx-phages would behave similarly to λ phage. However, contrary to the λ superinfection immunity model, it has been demonstrated that double lysogens can be produced with the Stx-phage Φ24B. Here, the Φ24B integrase gene is identified, and the preferred site of integration defined. Although an E. coli int gene was identified close to the Φ24B integration site, it was shown not to be involved in the phage integration event. An additional six potential integration sites were identified in the E. coli genome, and three of these were confirmed experimentally. Two of the other potential sites lie within genes predicted to be essential to E. coli and are therefore unlikely to support phage integration. A Φ24B gene, possessing similarity to the well-characterized P22 ant gene, was identified. RT-PCR was used to demonstrate that ant is transcribed in a Φ24B E. coli lysogen, and expression of an anti-repressor is the likely explanation for the absence of immunity to superinfection. Demonstration of the ability of Φ24B to form multiple lysogens has two potentially serious impacts. First, multiple integrated prophages will drive the evolution of bacterial pathogens as novel Stx-phages emerge following intracellular mutation/recombination events. Second, multiple copies of the stx gene may lead to an increase in toxin production and consequently increased virulence.
-
-
-
Organization of the biosynthetic gene cluster for the macrolide antibiotic spiramycin in Streptomyces ambofaciens
Spiramycin, a 16-membered macrolide antibiotic used in human medicine, is produced by Streptomyces ambofaciens; it comprises a polyketide lactone, platenolide, to which three deoxyhexose sugars are attached. In order to characterize the gene cluster governing the biosynthesis of spiramycin, several overlapping cosmids were isolated from an S. ambofaciens gene library, by hybridization with various probes (spiramycin resistance or biosynthetic genes, tylosin biosynthetic genes), and the sequences of their inserts were determined. Sequence analysis showed that the spiramycin biosynthetic gene cluster spanned a region of over 85 kb of contiguous DNA. In addition to the five previously described genes that encode the type I polyketide synthase involved in platenolide biosynthesis, 45 other genes have been identified. It was possible to propose a function for most of the inferred proteins in spiramycin biosynthesis, in its regulation, in resistance to the produced antibiotic or in the provision of extender units for the polyketide synthase. Two of these genes, predicted to be involved in deoxysugar biosynthesis, were inactivated by gene replacement, and the resulting mutants were unable to produce spiramycin, thus confirming their involvement in spiramycin biosynthesis. This work reveals the main features of spiramycin biosynthesis and constitutes a first step towards a detailed molecular analysis of the production of this medically important antibiotic.
-
-
-
CsoR regulates the copper efflux operon copZA in Bacillus subtilis
More LessThe adaptation of Bacillus subtilis to elevated levels of copper ions requires the copper-inducible copZA operon encoding a copper chaperone and efflux ATPase. Here we identify CsoR (formerly YvgZ) as the copper-sensing repressor that regulates the copZA operon. CsoR binds with high affinity to an operator site overlapping the copZA promoter and its binding is specifically inhibited by copper salts. As previously described, the YhdQ (CueR) protein also binds to the copZA regulatory region, but genetic experiments indicate that this protein is not responsible for the copper-dependent regulation of this operon.
-
-
-
VmeAB, an RND-type multidrug efflux transporter in Vibrio parahaemolyticus
Genes vmeA and vmeB, encoding a multidrug efflux transporter in the halophilic bacterium Vibrio parahaemolyticus, have been cloned using a drug-hypersusceptible Escherichia coli strain as the host. Cells of E. coli KAM33 (ΔacrAB ΔydhE) carrying the vmeAB region from V. parahaemolyticus conferred much higher MICs for a variety of antimicrobial agents than did control cells. Cells possessing VmeAB under energized conditions maintained very low intracellular concentrations of ethidium. This was as expected for an energy-dependent efflux system, and supports the notion – based on sequence homology – that VmeAB belongs to the resistance nodulation cell division (RND) family of multidrug efflux transporters. It is likely that VmeAB forms functional complexes with the outer-membrane protein TolC in E. coli, because introduction of vmeAB into cells of E. coli KAM43, which lacks the tolC gene, failed to elevate the MICs for any of the antimicrobial agents tested. Therefore, a V. parahaemolyticus homologue of tolC was also cloned, designated vpoC, and was introduced together with vmeAB into cells of E. coli KAM43. The MICs of all agents tested were raised and were comparable to the values observed in E. coli KAM33 harbouring a plasmid carrying vmeAB. Finally, a vmeAB-deficient mutant of V. parahaemolyticus was constructed (designated TM3). TM3 showed slightly higher susceptibility than the parental V. parahaemolyticus to some antimicrobial agents. Survival rate of the TM3 when exposed to deoxycholate decreased compared with that of the parent.
-
-
-
Comparative analysis of FimB and FimE recombinase activity
FimB and FimE are site-specific recombinases, part of the λ integrase family, and invert a 314 bp DNA switch that controls the expression of type 1 fimbriae in Escherichia coli. FimB and FimE differ in their activity towards the fim switch, with FimB catalysing inversion in both directions in comparison to the higher-frequency but unidirectional on-to-off recombination catalysed by FimE. Previous work has demonstrated that FimB, but not FimE, recombination is completely inhibited in vitro and in vivo by a regulator, PapB, expressed from a distinct fimbrial locus. The aim of this work was to investigate differences between FimB and FimE activity by exploiting the differential inhibition demonstrated by PapB. The research focused on genetic changes to the fim switch that alter recombinase binding and its structural context. FimB and FimE still recombined a switch in which the majority of fimS DNA was replaced with a larger region of non-fim DNA. This demonstrated a minimal requirement for FimB and FimE recombination of the Fim binding sites and associated inverted repeats. With the original leucine-responsive regulatory protein (Lrp) and integration host factor (IHF)-dependent structure removed, PapB was now able to inhibit both recombinases. The relative affinities of FimB and FimE were determined for the four ‘half sites’. This analysis, along with the effect of extensive swaps and duplications of the half sites on recombination frequency, demonstrated that FimB recruitment and therefore subsequent activity was dependent on a single half site and its context, whereas FimE recombination was less stringent, being able to interact initially with two half sites with equally high affinity. While increasing FimB recombination frequencies failed to overcome PapB repression, mutations made in recombinase binding sites resulted in inhibition of FimE recombination by PapB. Overall, the data support a model in which the recombinases differ in loading order and co-operative interactions. PapB exploits this difference and FimE becomes susceptible when its normal loading is restricted or changed.
-
-
-
Characterization of a Giardia lamblia WB C6 clone resistant to the isoflavone formononetin
More LessGiardia lamblia is a common intestinal-dwelling protozoan and causes diarrhoea in humans and animals worldwide. For several years, a small number of drugs such as the 5-nitroimidazole metronidazole (MET) or the thiazolide nitazoxanide (NTZ) have been used for chemotherapy against giardiasis. However, various pre-clinical and clinical investigations revealed that antigiardial chemotherapy may be complicated by emergence of giardial resistance to these drugs. The present study addressed the question if isoflavones with antigiardial activity, such as daidzein (DAI) or formononetin (FOR), may serve as alternative compounds for treatment of giardiasis. For this purpose, the potential of G. lamblia clone WB C6 to form resistance to FOR and related isoflavones was tested in vitro. In the line of these experiments, a clone (C3) resistant to isoflavones, but sensitive to MET and NTZ, was generated. Affinity chromatography on DAI-agarose using cell-free extracts of G. lamblia trophozoites resulted in the isolation of a polypeptide of approximately 40 kDa, which was identified by mass spectrometry as a nucleoside hydrolase (NH) homologue (EAA37551.1). In a nucleoside hydrolase assay, recombinant NH hydrolysed all nucleosides with a preference for purine nucleosides and was inhibited by isoflavones. Using quantitative RT-PCR, the expression of genes that are potentially involved in resistance formation was analysed, namely NH and genes encoding variant surface proteins (VSPs, TSA417). The transcript level of the potential target NH was found to be significantly reduced in C3. Moreover, drastic changes were observed in VSP gene expression. This may indicate that resistance formation in Giardia against isoflavones is linked to, and possibly mediated by, altered gene expression. Taken together, our results suggest FOR or related isoflavones as an alternative antigiardial agent to overcome potential problems of resistance to drugs like MET or NTZ. However, the capacity of Giardia to develop resistance to isoflavones can potentially interfere with this alternative treatment of the disease.
-
-
-
Analysis of the structure of mycolic acids of Mycobacterium simiae reveals a particular composition of α-mycolates in strain ‘habana’ TMC 5135, considered as immunogenic in tuberculosis and leprosy
More LessStructural analysis of mycolic acids from Mycobacterium simiae (including some ‘habana’ strains) was carried out using 1H-NMR and MS. Results indicated that this species presents a general pattern of α-, α′- and keto-mycolates. α-Mycolates were composed of a complex mixture of 82 to 89 carbon atoms (C82–C89), with the predominant molecular species containing two di-substituted cyclopropane rings. Among keto-mycolates (C84–C89), those containing one trans di-substituted cyclopropane ring were the most abundant. The α′-mycolates were monounsaturated (C64, C66). According to MS and 1H-NMR data, the strains studied differed in fine structural details of α-mycolates and keto-mycolates. Notably, strain ‘habana’ TMC 5135 (belonging to the ‘habana’ group, and considered as highly immunogenic in tuberculosis and leprosy) presented a particular composition of α-mycolates, with a major component (C87) containing one cis plus one trans di-substituted cyclopropane ring, unlike the type strain of M. simiae and other strains of the ‘habana’ group (IPK-220 and IPK-337R), in which the major component (C84) contained two cis di-substituted cyclopropane rings. In spite of this finding, the ‘habana’ strains were closely related to each other and mainly differed from the type strain of M. simiae in some details of the fine structure of keto-mycolates. The present work indicated that within an identical general pattern of mycolic acids, there is a complex composition in M. simiae and structural variation among different strains, as reported for pathogenic species of the genus. Noteworthy was the particular composition of α-mycolates in strain ‘habana’ TMC 5135.
-
-
-
Identification of the dehydratase component of the mycobacterial mycolic acid-synthesizing fatty acid synthase-II complex
More LessMycolic acids are vital components of the Mycobacterium tuberculosis cell wall and are essential for survival. While most components of the fatty acid synthase-II (FAS-II) enzymic machinery that synthesizes these long chain α-alkyl, β-hydroxy fatty acids have been identified, the gene encoding the β-hydroxyacyl-acyl carrier protein (ACP) dehydratase activity has remained elusive. Recent bioinformatics-based studies and drug inhibition experiments have identified the M. tuberculosis gene Rv0636 as a promising candidate for this role. Using a recently described, specialized transduction-based genetic tool we now demonstrate that MSMEG1341, the Mycobacterium smegmatis homologue of Rv0636, is an essential gene; null mutants of the gene could only be generated in a merodiploid strain which contained a second integrated acetamide-inducible copy of MSMEG1341. Growth of the conditional mutant in the absence of acetamide resulted in loss of mycolic acid biosynthesis and eventually loss of viability due to cell lysis. Null MSMEG1341 mutants could also be generated in a M. smegmatis strain containing an integrated copy of Rv0636, indicating that Rv0636 was the functional counterpart of MSMEG1341 in M. tuberculosis. Our results demonstrate that MSMEG1341 is an essential gene involved in mycolic acid biosynthesis and encodes the FAS-II β-hydroxyacyl-ACP dehydratase.
-
- Biodiversity And Evolution
-
-
-
Development of a multilocus sequence typing scheme for intestinal spirochaetes within the genus Brachyspira
The purpose of this study was to evaluate a multilocus sequence typing (MLST) scheme for intestinal spirochaetes of the genus Brachyspira. Eight loci mainly coding for enzymes previously used in multilocus enzyme electrophoresis analysis of Brachyspira species were examined in 66 Brachyspira field isolates and type/reference strains. The isolates and strains were recovered from pigs, birds, dogs and a mouse and originated from seven European countries, the USA and Canada. Forty-six isolates represented recognized Brachyspira species and 20 represented provisionally designated species or isolates that have not been classified. Only two loci gave PCR products for all 66 strains and isolates, but amplicons for seven loci were obtained for 44 of the isolates. Sequences for each locus had a DNA allelic variation of 30–47 and an amino acid allelic variation of 14–47 that gave rise to the same number of sequence and amino acid types (58) for the strains and isolates studied. A population snapshot based on sequence and amino acid types showed a close phylogenetic relationship amongst the porcine isolates from the same geographical regions, and indicated a close evolutionary relationship between isolates recovered from pigs and mallards. A general concordance was obtained between the MLST groupings and classifications based on culture and biochemical tests, 16S rDNA sequence analysis and random amplified polymorphic DNA analysis. This is a first step towards establishing an MLST system for use in identifying Brachyspira species and determining relationships between individual strains and species in the genus.
-
-
-
-
Protein expression diversity amongst serovars of Salmonella enterica
More LessSalmonella enterica is one of the most extensively studied bacterial species in terms of physiology, genetics, cell culture and development. As a very diverse group, the serovars of S. enterica display a spectrum of host specificities ranging from a broad host range to strictly host-adapted variants. This study utilized a classic proteomic approach combining 2D gel electrophoresis and mass spectrometry for the comparative analysis of the proteomes of serovars Typhimurium, Enteritidis, Choleraesuis, Pullorum and Dublin. The comparative analysis revealed species-specific protein factors with no significant change in expression amongst all isolates, as well as proteins with fluctuating expression levels between serovars and strains. Examples include an isoform of SodA specific for serovar Typhimurium, the third isoform of the lysine arginine ornithine (LAO)-binding amino acid transporter specific for serovar Pullorum, and the enzyme GabD found to be unique to serovar Choleraesuis. Overall the study demonstrated the importance of using multiple isolates when characterizing the expression patterns of bacteria in order to account for the intrinsic diversity of a bacterial population and revealed several factors with potential roles in host adaptation and pathogenicity of the serovars of S. enterica.
-
- Environmental Microbiology
-
-
-
Structure and function of the microbial community in a full-scale enhanced biological phosphorus removal plant
More LessThe structure and function of the microbial community in a full-scale enhanced biological phosphorus removal wastewater treatment plant (WWTP; Skagen) were investigated using the full-cycle rRNA approach, combined with ecophysiological studies. A total of 87 16S rRNA gene sequences were retrieved, and 78 operational taxonomic units were identified. Novel oligonucleotide probes were designed, and quantitative fluorescence in situ hybridization revealed that six hitherto undescribed probe-defined groups within the phylum Bacteroidetes (two groups), and classes Betaproteobacteria (two groups) and Gammaproteobacteria (two groups), were relatively abundant (>1 % of total biovolume) in the Skagen WWTP and 10 other full-scale WWTPs with biological P removal. The most abundant was a group of rod-shaped Bacteroidetes attached to filamentous bacteria, which is distantly related to the genus Haliscomenobacter of the family Saprospiraceae, and comprised 9–19 % of the bacterial biovolume in all the WWTPs investigated. The other five probe-defined groups were found in all WWTPs, but they were less abundant (1–6 %). Two groups had a glycogen-accumulating phenotype and one Dechloromonas-related group had a polyphosphate-accumulating phenotype, and they were potentially all involved in denitrification. In total, about 81 % of all bacteria hybridizing with the general eubacterial probe were detected in the Skagen WWTP by using clone- or group-specific probes, indicating that most members of the microbial community had been identified.
-
-
- Genes And Genomes
-
-
-
Cloning, characterization and expression of a gene encoding dihydroxyacetone synthase in Mycobacterium sp. strain JC1 DSM 3803
Dihydroxyacetone synthase (DHAS) is a key enzyme involved in the assimilation of methanol in Mycobacterium sp. strain JC1 DSM 3803. The structural gene encoding DHAS in Mycobacterium sp. strain JC1 was cloned using random-primed probes synthesized after PCR with synthetic primers based on the amino acid sequences conserved in two yeast DHASs and several transketolases. The cloned gene, dasS, had an ORF of 2193 nt, encoding a protein with a calculated molecular mass of 78 197 Da. The deduced amino acid sequence of dasS contained an internal sequence of Mycobacterium sp. strain JC1 DHAS and exhibited 29.2 and 27.3 % identity with those of Candida boidinii and Hansenula polymorpha enzymes, respectively. Escherichia coli transformed with the cloned gene produced a novel protein with a molecular mass of ∼78 kDa, which cross-reacted with anti-DHAS antiserum and exhibited DHAS activity. Primer-extension analysis revealed that the transcriptional start site of the gene was the nucleotide A located 31 bp upstream from the dasS start codon. RT-PCR showed that dasS was transcribed as a monocistronic message. Northern hybridization and β-galactosidase assay with the putative promoter region of dasS revealed that the gene was transcribed only in cells growing on methanol. The expression of dasS in Mycobacterium sp. strain JC1 was free from catabolite repression.
-
-
-
-
Transcriptome profiling of Paracoccidioides brasiliensis yeast-phase cells recovered from infected mice brings new insights into fungal response upon host interaction
Milce Costa, Clayton L. Borges, Alexandre M. Bailão, Gabriela V. Meirelles, Yuri A. Mendonça, Sabrina F. I. M. Dantas, Fabrícia P. de Faria, Maria S. S. Felipe, Eugênia E. W. I. Molinari-Madlum, Maria J. S. Mendes-Giannini, Rogério B. Fiuza, Wellington S. Martins, Maristela Pereira and Célia M. A. SoaresParacoccidioides brasiliensis is a fungal human pathogen with a wide distribution in Latin America. It causes paracoccidioidomycosis, the most widespread systemic mycosis in Latin America. Although gene expression in P. brasiliensis had been studied, little is known about the genome sequences expressed by this species during the infection process. To better understand the infection process, 4934 expressed sequence tags (ESTs) derived from a non-normalized cDNA library from P. brasiliensis (isolate Pb01) yeast-phase cells recovered from the livers of infected mice were annotated and clustered to a UniGene (clusters containing sequences that represent a unique gene) set with 1602 members. A large-scale comparative analysis was performed between the UniGene sequences of P. brasiliensis yeast-phase cells recovered from infected mice and a database constructed with sequences of the yeast-phase and mycelium transcriptome (isolate Pb01) (https://dna.biomol.unb.br/Pb/), as well as with all public ESTs available at GenBank, including sequences of the P. brasiliensis yeast-phase transcriptome (isolate Pb18) (http://www.ncbi.nlm.nih.gov/). The focus was on the overexpressed and novel genes. From the total, 3184 ESTs (64.53 %) were also present in the previously described transcriptome of yeast-form and mycelium cells obtained from in vitro cultures (https://dna.biomol.unb.br/Pb/) and of those, 1172 ESTs (23.75 % of the described sequences) represented transcripts overexpressed during the infection process. Comparative analysis identified 1750 ESTs (35.47 % of the total), comprising 649 UniGene sequences representing novel transcripts of P. brasiliensis, not previously described for this isolate or for other isolates in public databases. KEGG pathway mapping showed that the novel and overexpressed transcripts represented standard metabolic pathways, including glycolysis, amino acid biosynthesis, lipid and sterol metabolism. The unique and divergent representation of transcripts in the cDNA library of yeast cells recovered from infected mice suggests differential gene expression in response to the host milieu.
-
- Pathogens And Pathogenicity
-
-
-
Cryptococcus neoformans laccase catalyses melanin synthesis from both d- and l-DOPA
The human fungal pathogen Cryptococcus neoformans produces melanin in the presence of various substrates, including the l enantiomer of 3,4-dihydroxyphenylalanine (DOPA). The enzyme laccase catalyses the formation of melanin by oxidizing l-DOPA, initiating a series of presumably spontaneous reactions that ultimately leads to the polymerization of the pigment in the yeast cell wall. There, melanin protects the cell from a multitude of environmental and host assaults. Thus, the ability of C. neoformans to produce pigments from a variety of available substrates is likely to confer a survival advantage. A number of C. neoformans isolates of different serotypes produced pigments from d-DOPA, the stereoisomer of l-DOPA. Acid-resistant particles were isolated from pigmented C. neoformans cells grown in the presence of d-DOPA. Biophysical characterization showed the particles had a stably detectable free-radical signal by EPR, and negative zeta potential, similar to l-DOPA-derived particles. No major differences were found between l- and d-DOPA ghosts in terms of binding to anti-melanin antibodies, or in overall architecture when imaged by electron microscopy. C. neoformans cells utilized l- and d-DOPA at a similar rate. Overall, our results indicate that C. neoformans shows little stereoselectivity for utilizing DOPA in melanin synthesis. The ability of C. neoformans to use both l and d enantiomers for melanization implies that this organism has access to a greater potential pool of substrates for melanin synthesis, and this could potentially be exploited in the design of therapeutic inhibitors of laccase.
-
-
-
-
The group B streptococcal alpha C protein binds α 1 β 1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells
More LessGroup B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis and meningitis among neonates and a cause of morbidity among pregnant women and immunocompromised adults. GBS epithelial cell invasion is associated with expression of alpha C protein (ACP). Loss of ACP expression results in a decrease in GBS internalization and translocation across human cervical epithelial cells (ME180). Soluble ACP and its 170 amino acid N-terminal region (NtACP), but not the repeat protein RR′, bind to ME180 cells and reduce internalization of wild-type GBS to levels obtained with an ACP-deficient isogenic mutant. In the current study, ACP colocalized with α 1 β 1-integrin, resulting in integrin clustering as determined by laser scanning confocal microscopy. NtACP contains two structural domains, D1 and D2. D1 is structurally similar to fibronectin's integrin-binding region (FnIII10). D1's (KT)D146 motif is structurally similar to the FnIII10 (RG)D1495 integrin-binding motif, suggesting that ACP binds α 1 β 1-integrin via the D1 domain. The (KT)D146A mutation within soluble NtACP reduced its ability to bind α 1 β 1-integrin and inhibit GBS internalization within ME180 cells. Thus ACP binding to human epithelial cell integrins appears to contribute to GBS internalization within epithelial cells.
-
-
-
SufA – a novel subtilisin-like serine proteinase of Finegoldia magna
Finegoldia magna is an anaerobic Gram-positive bacterium and commensal, which is also associated with clinically important conditions such as skin and soft tissue infections. This study describes a novel subtilisin-like extracellular serine proteinase of F. magna, denoted SufA (subtilase of Finegoldia magna ), which is believed to be the first subtilase described among Gram-positive anaerobic cocci. SufA is associated with the bacterial cell surface, but is also released in substantial amounts during bacterial growth. Papain was used to release SufA from the surface of F. magna and the enzyme was purified by ion-exchange chromatography and gel filtration. A protein band on SDS-PAGE corresponding to the dominating proteolytic activity on gelatin zymography was analysed by MS/MS. Based on the peptide sequences obtained, the sufA gene was sequenced. The gene comprises 3466 bp corresponding to a preprotein of 127 kDa. Like other members of the subtilase family, SufA contains the catalytic triad of aspartic acid, histidine and serine with surrounding conserved residues. A SufA homologue was identified in 33 of 34 investigated isolates of F. magna, as revealed by PCR and immunoprinting. The enzyme forms dimers, which are more proteolytically active than the monomeric protein. SufA was found to efficiently cleave and inactivate the antibacterial peptide LL-37 and the CXC chemokine MIG/CXCL9, indicating that the enzyme promotes F. magna survival and colonization.
-
-
-
Regulation of the Pseudomonas aeruginosa toxA, regA and ptxR genes by the iron-starvation sigma factor PvdS under reduced levels of oxygen
The level of environmental oxygen (EO) within various Pseudomonas aeruginosa infection sites is low (microaerobic), and this can affect the production of different virulence factors. Expression of the toxA gene, encoding exotoxin A (ETA), is regulated by regA, ptxR and pvdS. Moreover, the iron-starvation sigma factor PvdS directs the transcription of pyoverdine siderophore genes (e.g. pvdD). DNA–protein binding analysis using recombinant PvdS showed that the PvdS–RNA polymerase holoenzyme complex specifically bound the toxA, regA and ptxR promoter regions. All three promoters contain a PvdS-binding site, the iron-starvation box. To determine the relationship between these different genes and PvdS, we conducted a comparative analysis of toxA, regA, ptxR and pvdD transcription throughout the growth cycle of wild-type P. aeruginosa and its pvdS mutant in iron-deficient medium under aerobic-shaking (A-sh) and microaerobic-static (M-st) conditions. Under both EO conditions, optimal toxA, regA and pvdD expression and pyoverdine production required PvdS, while ptxR expression was moderately dependent on PvdS only under A-sh conditions. Expression of regA, pvdD and pyoverdine production in wild-type P. aeruginosa was significantly lower under M-st in comparison with A-sh conditions, while the opposite was observed for toxA and ptxR. Although low, the level of toxA expression and ETA production in the pvdS mutant were higher under M-st than under A-sh conditions. Transcription of pvdS and PvdS expression were also reduced by low EO. We propose that the regulation of toxA expression under aerobic conditions primarily involves PvdS, while an additional EO-responsive regulator(s) besides PvdS is required under low EO levels. Thus, PvdS may control the transcription of the ptxR, regA and toxA genes, and respond to EO by acting at different levels of the toxA regulatory cascade.
-
-
-
The stringent response of Bacillus anthracis contributes to sporulation but not to virulence
More LessThe Gram-positive, spore-forming pathogen Bacillus anthracis is the aetiological agent of anthrax. Its main virulence factors are two toxins and an anti-phagocytic capsule. When B. anthracis is grown in laboratory culture, the highest expression of the anthrax toxin genes occurs during entry into stationary phase, suggesting that nutrient limitation is an environmental cue which induces toxin production. A common bacterial response to starvation is the so-called stringent response, in which the hyperphosphorylated guanosine nucleotide (p)ppGpp is the effector molecule. In Escherichia coli, Bacillus subtilis and other bacteria, accumulation of this molecule leads to down-regulation of stable RNA synthesis and upregulation of the expression of genes involved in survival under nutrient-poor conditions. This study focuses on the stringent response of B. anthracis. We show that in B. anthracis the relA gene is responsible for the synthesis of (p)ppGpp and the stringent down-regulation of stable RNA synthesis upon starvation for the essential amino acids isoleucine, leucine and valine. The deletion of relA did not affect the expression of the virulence gene pagA or virulence in a mouse model of infection. In contrast, spore counts upon growth and sporulation in a defined medium were approximately 10 000-fold lower for the relA deletion mutant than for the parental strain. The contribution of the stringent response to efficient sporulation of B. anthracis is notable, as this suggests that the stringent response may contribute to the persistence of B. anthracis in the natural environment.
-
-
-
Invasion of HeLa cells by group B streptococcus requires the phosphoinositide-3-kinase signalling pathway and modulates phosphorylation of host-cell Akt and glycogen synthase kinase-3
More LessThe group B streptococcus (GBS) is an opportunistic bacterial pathogen with the ability to cause invasive disease. While the ability of GBS to invade a number of host-cell types has been clearly demonstrated, the invasion process is not well understood at the molecular level. What has been well established is that modulation of host-cell actin microfilaments is essential for GBS invasion to occur. Phosphoinositide-3 kinase (PI3K) is a key regulator of the cytoskeleton in eukaryotic cells. Our goal in this investigation was to explore the role of the PI3K/Akt signalling pathway in epithelial cell invasion by GBS. The epithelial cell invasion process was mimicked using the HeLa 229 cell-culture model. Treating HeLa cells with chemical inhibitors of PI3K, Akt or Ras prior to bacterial infection inhibited GBS invasion but not attachment; treatment with 30 μM LY294002 (PI3K inhibitor) reduced GBS invasion by 75 %, 20 μM l-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (ICIO) (Akt inhibitor) reduced GBS invasion by 50 %, and 10 μM manumycin A (Ras inhibitor) inhibited GBS invasion by 90 %. Genetic inactivation of the p85α or p110α PI3K subunits in HeLa cells also reduced GBS invasion by 55 and 30 %, respectively. Western blot analysis revealed that phosphorylation of host-cell Akt and glycogen synthase kinase-3 (GSK-3) occurs in response to GBS infection, and that this is mediated upstream by PI3K. Infection of HeLa cells with GBS triggers pro-survival signalling and protects the HeLa cells from camptothecin-induced caspase-3 cleavage. The results from this investigation show that GBS both requires and activates the PI3K/Akt host-cell signalling pathway during invasion of epithelial cells.
-
-
-
Characterization of the NAD-glycohydrolase in streptococcal strains
The NADase (Nga) of group A streptococci (GAS) has been implicated in the pathogenesis of diseases such as streptococcal toxic shock-like syndrome (STSS) and necrotizing fasciitis. In this study we found that the proportion of NADase-positive strains among clinical isolates in Japan has increased over time. The GAS strains studied could be divided into three groups: strains lacking NADase activity, strains with low NADase activity, and strains with high NADase activity. The older strains, isolated before 1989, belonged to the ‘no activity’ group. Analysis using GST–Nga recombinants revealed that nga alleles of representative older strains encode inactive Nga. Mutational analysis of the GST–Nga recombinants suggested that residue 330 could be associated with reduced activity, based upon deduced amino acid sequences. We also investigated NADase activity of streptococcal strains other than GAS. All group G streptococcal isolates from STSS patients possessed nga genes encoding active enzymes.
-
- Physiology
-
-
-
Characterization of the ferrioxamine uptake system of Nitrosomonas europaea
More LessThe chemolithoautotroph Nitrosomonas europaea has two genes predicted to encode outer-membrane (OM) ferrioxamine transporters. Expression of the ferrioxamine uptake system required induction, as shown by the shorter lag phase in ferrioxamine-containing cultures when ferrioxamine-exposed cells were used as an inoculum. The two OM ferrioxamine siderophore transporters encoded by foxA1 (NE1097) and foxA2 (NE1088) were produced only in cells grown in Fe-limited ferrioxamine-containing medium. The inactivation of foxA1 , singly or in combination with foxA2 , prevented growth in Fe-limited medium containing excess desferrioxamine (DFX). The foxA2 -disrupted single mutant grew poorly in the regular Fe-limited (0.2 μM) medium with 10 μM DFX, but grew well when the Fe level was raised to 1.0 μM with 10 μM DFX. For efficient acquisition of Fe-loaded ferrioxamine, N. europaea needs both ferrioxamine transporters FoxA1 and FoxA2. FoxA1 probably regulates its own production, and it controls the production of FoxA2 as well.
-
-
-
-
Osmotic adaptation of the halophilic fungus Hortaea werneckii: role of osmolytes and melanization
This study was intended to determine the osmoadaptation strategy of Hortaea werneckii, an extremely salt-tolerant melanized ascomycetous fungus that can grow at 0–5.1 M NaCl. It has been shown previously that glycerol is the major compatible solute in actively growing H. werneckii. This study showed that the exponentially growing cells also contained erythritol, arabitol and mannitol at optimal growth salinities, but only glycerol and erythritol at maximal salinities. The latter two were both demonstrated to be major compatible solutes in H. werneckii, as their decrease correlated with the severity of hypoosmotic shock. Besides higher amounts of erythritol and lower amounts of glycerol, stationary-phase cells also contained mycosporine-glutaminol-glucoside, which might act as a complementary compatible solute. H. werneckii is constitutively melanized under various salinity conditions. Ultrastructural study showed localization of melanin in the outer parts of the cell wall as a distinct layer at optimal salinity (0.86 M NaCl), whereas cell-wall melanization diminished at higher salinities. The role of melanized cell wall in the effective retention of glycerol is already known, and was also demonstrated in H. werneckii by lower retention of glycerol in cells with blocked melanization compared to melanized cells. However, these non-melanized cells compensated for the lower amounts of glycerol with higher amounts of erythritol and arabitol. We hypothesize that H. werneckii melanization is effective in reducing the permeability of its cell wall to its major compatible solute glycerol, which might be one of the features that helps it tolerate a wider range of salt concentrations than most organisms.
-
-
-
Molecular characterization of the copper transport system in Staphylococcus aureus
More LessThe Staphylococcus aureus copA gene codes for a putative copper-translocating P-type ATPase and the downstream copZ gene codes for a copper chaperone. Genome database analyses demonstrate that these copper transport genes are highly conserved in S. aureus. The expression of copA and copZ was inducible by copper and to some extent by ferric and lead ions. A mutant strain containing a partially deleted copA gene was more sensitive than the parent strain to copper, ferric and lead ions. The copper-sensitive phenotype was due to the accumulation of intracellular copper and thus the copA product is involved in the export of copper ions. The metal-sensitive phenotype of the mutant was complemented in trans by a 2.7 kbp DNA containing copA. We have cloned and overexpressed the metal-binding domains of CopA and CopZ and have shown by site-directed mutagenesis that the cysteine residues in the CXXC metal-binding motif in CopA are involved in copper binding and thus play an important role in copper transport in S. aureus.
-
- Plant-Microbe Interactions
-
-
-
A Mesorhizobium loti mutant with reduced glucan content shows defective invasion of its host plant Lotus japonicus
More LessRandom transposon mutagenesis led to the isolation of a novel Mesorhizobium loti mutant that is defective in nitrogen fixation during symbiosis with Lotus japonicus. The mutated locus, designated cep, encodes a putative cell-envelope protein displaying no significant sequence similarity to proteins with known functions. This mutant elicits the formation of nodule-like bumps and root-hair curling, but not the elongation of infection threads, on L. japonicus roots. This is reminiscent of the phenotypes of rhizobial mutants impaired in cyclic β-glucan biosynthesis. The cep mutant exhibits partially reduced content of cell-associated glucans and intermediate deficiency of motility under hypo-osmotic conditions as compared to a glucan-deficient mutant. Second-site pseudorevertants of the cep mutant were isolated by selecting for restoration of symbiotic nitrogen fixation. A subset of pseudorevertants restored both symbiotic capability and glucan content to levels comparable to that of the wild-type. These results suggest that the Cep product acts on a successful symbiosis by affecting cell-associated glucan content.
-
-
-
-
The role of glucose kinase in carbohydrate utilization and extracellular polysaccharide production in Xanthomonas campestris pathovar campestris
The genome of the Xanthomonas campestris pathovar campestris (Xcc) strain 8004 encodes three uncharacterized proteins, XC1166, XC1223 and XC1976, annotated as glucose kinase (Glk) by bioinformatic studies. Here we have investigated the biochemical characteristics and physiological roles of these proteins with particular reference to the synthesis of extracellular polysaccharide (EPS). XC1166, XC1223 and XC1976 were overexpressed as fusion proteins with a His6 affinity tag and purified by nickel affinity chromatography. The standard Glk activity assay revealed that all three proteins possessed apparent Glk activity, with XC1976-His6 being the most active; the specific activity values were 1.16×106 U mg−1 for XC1166-His6, 4.36×107 U mg−1 for XC1223-His6 and 2.63×108 U mg−1 for XC1976-His6. TLC analysis showed, however, that only XC1976-His6 could phosphorylate glucose. Insertional mutants of XC1166, XC1223 and XC1976 were generated using the suicide plasmid pK18mob. Although mutant strains with insertions in XC1166 or XC1223 had Glk activity similar to that of the wild-type strain, the XC1976 mutant had only about 6 % of the wild-type activity. Mutation in XC1976 had complex effects on EPS production. In media containing arabinose, glucose, galactose, sucrose or maltose, the XC1976 mutant produced about 40–75 % of the wild-type level of EPS, whereas in medium containing fructose, the mutant showed a 30 % increase in EPS production compared to the wild-type strain. The XC1976 mutant also showed attenuated virulence on the host plant Chinese radish (Raphanus sativus). The results indicate that XC1976 has the most significant role for the parameters tested.
-
Volumes and issues
-
Volume 170 (2024)
-
Volume 169 (2023)
-
Volume 168 (2022)
-
Volume 167 (2021)
-
Volume 166 (2020)
-
Volume 165 (2019)
-
Volume 164 (2018)
-
Volume 163 (2017)
-
Volume 162 (2016)
-
Volume 161 (2015)
-
Volume 160 (2014)
-
Volume 159 (2013)
-
Volume 158 (2012)
-
Volume 157 (2011)
-
Volume 156 (2010)
-
Volume 155 (2009)
-
Volume 154 (2008)
-
Volume 153 (2007)
-
Volume 152 (2006)
-
Volume 151 (2005)
-
Volume 150 (2004)
-
Volume 149 (2003)
-
Volume 148 (2002)
-
Volume 147 (2001)
-
Volume 146 (2000)
-
Volume 145 (1999)
-
Volume 144 (1998)
-
Volume 143 (1997)
-
Volume 142 (1996)
-
Volume 141 (1995)
-
Volume 140 (1994)
-
Volume 139 (1993)
-
Volume 138 (1992)
-
Volume 137 (1991)
-
Volume 136 (1990)
-
Volume 135 (1989)
-
Volume 134 (1988)
-
Volume 133 (1987)
-
Volume 132 (1986)
-
Volume 131 (1985)
-
Volume 130 (1984)
-
Volume 129 (1983)
-
Volume 128 (1982)
-
Volume 127 (1981)
-
Volume 126 (1981)
-
Volume 125 (1981)
-
Volume 124 (1981)
-
Volume 123 (1981)
-
Volume 122 (1981)
-
Volume 121 (1980)
-
Volume 120 (1980)
-
Volume 119 (1980)
-
Volume 118 (1980)
-
Volume 117 (1980)
-
Volume 116 (1980)
-
Volume 115 (1979)
-
Volume 114 (1979)
-
Volume 113 (1979)
-
Volume 112 (1979)
-
Volume 111 (1979)
-
Volume 110 (1979)
-
Volume 109 (1978)
-
Volume 108 (1978)
-
Volume 107 (1978)
-
Volume 106 (1978)
-
Volume 105 (1978)
-
Volume 104 (1978)
-
Volume 103 (1977)
-
Volume 102 (1977)
-
Volume 101 (1977)
-
Volume 100 (1977)
-
Volume 99 (1977)
-
Volume 98 (1977)
-
Volume 97 (1976)
-
Volume 96 (1976)
-
Volume 95 (1976)
-
Volume 94 (1976)
-
Volume 93 (1976)
-
Volume 92 (1976)
-
Volume 91 (1975)
-
Volume 90 (1975)
-
Volume 89 (1975)
-
Volume 88 (1975)
-
Volume 87 (1975)
-
Volume 86 (1975)
-
Volume 85 (1974)
-
Volume 84 (1974)
-
Volume 83 (1974)
-
Volume 82 (1974)
-
Volume 81 (1974)
-
Volume 80 (1974)
-
Volume 79 (1973)
-
Volume 78 (1973)
-
Volume 77 (1973)
-
Volume 76 (1973)
-
Volume 75 (1973)
-
Volume 74 (1973)
-
Volume 73 (1972)
-
Volume 72 (1972)
-
Volume 71 (1972)
-
Volume 70 (1972)
-
Volume 69 (1971)
-
Volume 68 (1971)
-
Volume 67 (1971)
-
Volume 66 (1971)
-
Volume 65 (1971)
-
Volume 64 (1970)
-
Volume 63 (1970)
-
Volume 62 (1970)
-
Volume 61 (1970)
-
Volume 60 (1970)
-
Volume 59 (1969)
-
Volume 58 (1969)
-
Volume 57 (1969)
-
Volume 56 (1969)
-
Volume 55 (1969)
-
Volume 54 (1968)
-
Volume 53 (1968)
-
Volume 52 (1968)
-
Volume 51 (1968)
-
Volume 50 (1968)
-
Volume 49 (1967)
-
Volume 48 (1967)
-
Volume 47 (1967)
-
Volume 46 (1967)
-
Volume 45 (1966)
-
Volume 44 (1966)
-
Volume 43 (1966)
-
Volume 42 (1966)
-
Volume 41 (1965)
-
Volume 40 (1965)
-
Volume 39 (1965)
-
Volume 38 (1965)
-
Volume 37 (1964)
-
Volume 36 (1964)
-
Volume 35 (1964)
-
Volume 34 (1964)
-
Volume 33 (1963)
-
Volume 32 (1963)
-
Volume 31 (1963)
-
Volume 30 (1963)
-
Volume 29 (1962)
-
Volume 28 (1962)
-
Volume 27 (1962)
-
Volume 26 (1961)
-
Volume 25 (1961)
-
Volume 24 (1961)
-
Volume 23 (1960)
-
Volume 22 (1960)
-
Volume 21 (1959)
-
Volume 20 (1959)
-
Volume 19 (1958)
-
Volume 18 (1958)
-
Volume 17 (1957)
-
Volume 16 (1957)
-
Volume 15 (1956)
-
Volume 14 (1956)
-
Volume 13 (1955)
-
Volume 12 (1955)
-
Volume 11 (1954)
-
Volume 10 (1954)
-
Volume 9 (1953)
-
Volume 8 (1953)
-
Volume 7 (1952)
-
Volume 6 (1952)
-
Volume 5 (1951)
-
Volume 4 (1950)
-
Volume 3 (1949)
-
Volume 2 (1948)
-
Volume 1 (1947)