-
Volume 152,
Issue 9,
2006
Volume 152, Issue 9, 2006
- Review
-
-
-
Hypermutable bacteria isolated from humans – a critical analysis
More LessHypermutable bacteria of several species have been described among isolates recovered from humans over the last decade. Interpretation of the literature in this area is complicated by diversity in the determination and definition of hypermutability, and this review outlines the different methods used. Inactivation of the mismatch repair gene mutS is often implicated in the mutator phenotype; the reported effect of mutS inactivation on mutation frequency varies widely between species, from under 10-fold to nearly 1000-fold, but also varies among different reports on the same species. Particularly high proportions of mutators have been reported among Pseudomonas aeruginosa and other species in the cystic fibrosis lung, epidemic serogroup A Neisseria meningitidis, and Helicobacter pylori. Aspects of the biology of these infections that could be relevant to hypermutability are discussed, and some future directions that may increase our understanding of mutators among bacteria isolated from humans are considered.
-
-
-
-
Rho-dependent terminators and transcription termination
More LessRho-dependent transcription terminators participate in sophisticated genetic regulatory mechanisms, in both bacteria and phages; they occur in regulatory regions preceding the coding sequences of genes and within coding sequences, as well as at the end of transcriptional units, to prevent readthrough transcription. Most Rho-dependent terminators have been found in enteric bacteria, but they also occur in Gram-positive bacteria and may be widespread among bacteria. Rho-dependent termination requires both cis-acting elements, on the mRNA, and trans-acting factors. The only cis-acting element common to Rho-dependent terminators is richness in rC residues. Additional sequence elements have been observed at different Rho termination sites. These ‘auxiliary elements' may assist in the termination process; they differ among terminators, their occurrence possibly depending on the function and sequence context of the terminator. Specific nucleotides required for termination have also been identified at Rho sites. Rho is the main factor required for termination; it is a ring-shaped hexameric protein with ATPase and helicase activities. NusG, NusA and NusB are additional factors participating in the termination process. Rho-dependent termination occurs by binding of Rho to ribosome-free mRNA, C-rich sites being good candidates for binding. Rho's ATPase is activated by Rho–mRNA binding, and provides the energy for Rho translocation along the mRNA; translocation requires sliding of the message into the central hole of the hexamer. When a polymerase pause site is encountered, the actual termination occurs, and the transcript is released by Rho's helicase activity. Many aspects of this process are still being studied. The isolation of mutants suppressing termination, site-directed mutagenesis of cis-acting elements in Rho-dependent termination, and biochemistry, are and will be contributing to unravelling the still undefined aspects of the Rho termination machinery. Analysis of the more sophisticated regulatory mechanisms relying on Rho-dependent termination may be crucial in identifying new essential elements for termination.
-
- Biochemistry And Molecular Biology
-
-
-
Microdiesel: Escherichia coli engineered for fuel production
More LessBiodiesel is an alternative energy source and a substitute for petroleum-based diesel fuel. It is produced from renewable biomass by transesterification of triacylglycerols from plant oils, yielding monoalkyl esters of long-chain fatty acids with short-chain alcohols such as fatty acid methyl esters and fatty acid ethyl esters (FAEEs). Despite numerous environmental benefits, a broader use of biodiesel is hampered by the extensive acreage required for sufficient production of oilseed crops. Therefore, processes are urgently needed to enable biodiesel production from more readily available bulk plant materials like sugars or cellulose. Toward this goal, the authors established biosynthesis of biodiesel-adequate FAEEs, referred to as Microdiesel, in metabolically engineered Escherichia coli. This was achieved by heterologous expression in E. coli of the Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase and the unspecific acyltransferase from Acinetobacter baylyi strain ADP1. By this approach, ethanol formation was combined with subsequent esterification of the ethanol with the acyl moieties of coenzyme A thioesters of fatty acids if the cells were cultivated under aerobic conditions in the presence of glucose and oleic acid. Ethyl oleate was the major constituent of these FAEEs, with minor amounts of ethyl palmitate and ethyl palmitoleate. FAEE concentrations of 1.28 g l−1 and a FAEE content of the cells of 26 % of the cellular dry mass were achieved by fed-batch fermentation using renewable carbon sources. This novel approach might pave the way for industrial production of biodiesel equivalents from renewable resources by employing engineered micro-organisms, enabling a broader use of biodiesel-like fuels in the future.
-
-
-
-
A link in transcription between the native pbpB and the acquired mecA gene in a strain of Staphylococcus aureus
More LessConditional mutants of pbpB with an IPTG-inducible promoter were used to compare the effects of interrupted transcription of this gene in a meticillin-sensitive (MSSA) and a meticillin-resistant (MRSA) strain of Staphylococcus aureus. After 3 h growth following the removal of IPTG, multiplication of the MSSA strain stopped abruptly, cells began to lyse, and membrane preparations showed greatly decreased quantities of penicillin-binding protein (PBP) 2. In contrast, the MRSA strain continued to grow for at least 20 h in the IPTG-free medium, but with gradually increasing doubling times, which eventually reached 180 min. The peptidoglycan produced during this period of extremely slow growth showed only minor alterations, but cells with abnormal morphology accumulated in the culture, the abundance of mecA transcript gradually declined, and the cellular amounts of PBP2A were significantly decreased. Adding back the IPTG inducer caused rapid resumption in the transcription of pbpB, followed by an increase in the transcription of mecA. No changes were detected in the transcription of pbpA, C and D, the determinant of 16S rRNA or the housekeeping gene pta. Promoter fusion experiments suggested that the transcription of the resistance gene mecA may respond to some regulatory signal generated in the bacteria during changes in the transcription of pbpB.
-
-
-
The effect of penicillin on Chlamydia trachomatis DNA replication
More LessChlamydia trachomatis L2 was used to infect BGMK cells at an m.o.i. of 1.0, and the developmental cycle was followed by transmission electron microscopy and quantitative PCR (QPCR) for both chromosomal and plasmid DNA. Samples were taken at sequential 6 h time points. Subsequent analysis by QPCR showed that there was an initial slow replication period (0–18 h), followed by a rapid phase (18–36 h) coinciding with exponential division when the DNA doubling time was 4.6 h. Chromosomal DNA was amplified 100–200-fold corresponding to 7–8 generations for the complete developmental cycle. Penicillin (10 and 100 units ml−1) was added to cultures at 20 h post-infection (p.i.). This blocked binary fission and also prevented reticulate body (RB) to elementary body transition. However, exposure to penicillin did not prevent chromosomal or plasmid DNA replication. After a short lag period, following the addition of penicillin, chlamydial chromosomal DNA replication resumed at the same rate as in control C. trachomatis-infected cells. C. trachomatis-infected host cells exposed to penicillin did not lyse, but instead harboured large, aberrant RBs in massive inclusions that completely filled the cell cytoplasm. In these RBs, the DNA continued to replicate well beyond the end of the normal developmental cycle. At 60 h p.i. each aberrant RB contained a minimum of 16 chromosomal copies.
-
-
-
KlRHO1 and KlPKC1 are essential for cell integrity signalling in Kluyveromyces lactis
More LessCell integrity in yeasts is ensured by a rigid cell wall whose synthesis is triggered by a MAP kinase-mediated signal-transduction cascade. Upstream regulatory components of this pathway in Saccharomyces cerevisiae involve a single protein kinase C, which is regulated by interaction with the small GTPase Rho1. Here, two genes were isolated which encode these proteins from Kluyveromyces lactis (KlPKC1 and KlRHO1). Sequencing showed ORFs which encode proteins of 1161 and 208 amino acids, respectively. The deduced proteins shared 59 and 85 % overall amino acid identities, respectively, with their homologues from S. cerevisiae. Null mutants in both genes were non-viable, as shown by tetrad analyses of the heterozygous diploid strains. Overexpression of the KlRHO1 gene under the control of the ScGAL1 promoter severely impaired growth in both S. cerevisiae and K. lactis. On the other hand, a similar construct with KlPKC1 did not show a pronounced phenotype. Two-hybrid analyses showed interaction between Rho1 and Pkc1 for the K. lactis proteins and their S. cerevisiae homologues. A green fluorescent protein (GFP) fusion to the C-terminal end of KlPkc1 located the protein to patches in the growing bud, and at certain stages of the division process also to the bud neck. N-terminal GFP fusions to KlRho1 localized mainly to the cell surface (presumably the cytoplasmic side of the plasma membrane) and to the vacuole, with some indications of traffic from the former to the latter. Thus, KlPkc1 and KlRho1 have been shown to serve vital functions in K. lactis, to interact in cell integrity signalling and to traffic between the plasma membrane and the vacuole.
-
-
-
Cyt1Ca from Bacillus thuringiensis subsp. israelensis: production in Escherichia coli and comparison of its biological activities with those of other Cyt-like proteins
The larvicidal activity of Bacillus thuringiensis subsp. israelensis against dipteran larvae is determined by four major polypeptides of the parasporal crystalline body produced during sporulation. Cyt1Aa shows the lowest toxicity when used alone but is the most synergistic with any of the other proteins. The sequence of the plasmid pBtoxis, which contains all the toxin genes in this subspecies, revealed a new cyt-like coding sequence named cyt1Ca. In addition to the Cyt-like region, the predicted Cyt1Ca contained an extra domain at the C terminus, which appeared to be a β-trefoil carbohydrate-binding motif, as found in several ricin-like toxins. The gene was PCR-amplified from pBtoxis and cloned in several vectors, allowing high-level expression in Escherichia coli. Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae of Aedes aegypti of Cyt1Ca in recombinant E. coli cells was compared with that of Cyt1Aa and Cyt2Ba, and the ability of these proteins to enhance the activity of Cry4Aa was assessed. Although Cyt2Ba appeared able to interact with Cry4Aa, no activity for Cyt1Ca was observed, even when produced in truncated form. Furthermore, in contrast to Cyt1Aa, Cyt1Ca did not lyse sheep erythrocytes, and it was not bactericidal to the host cell.
-
-
-
Regulation of class D β-lactamase gene expression in Ralstonia pickettii
More LessRalstonia pickettii, an environmental bacterium that may also be responsible for human infections, produces two unrelated, inducible and chromosomally encoded oxacillinases, OXA-22 and OXA-60. In order to study the molecular basis of the induction process of these oxacillinase genes, the induction kinetics, the promoter/operator regions necessary for expression and induction, and the role of several ORFs located upstream and downstream of the bla OXA genes were investigated. The β-lactamase production reached a maximal level after 1 h induction, returned to its basal level within the following 3 h and was then again inducible. Using 5′RACE experiments, the promoter sequences of both oxacillinases were determined. These sequences showed weak promoter activities, which could, however, be increased approximately 200-fold by mutating the −35 promoter sequence. Deletion of the sequences located upstream of the promoter regions did not modify the basal β-lactamase expression in R. pickettii, but resulted in the lack of induction. A minimum of 240 and 270 bp upstream of the transcription initiation sites was required for inducible expression of the bla OXA-22 and bla OXA-60 genes, respectively. Analysis of the genetic environment of both bla OXA genes revealed several ORFs that were inactivated by homologous recombination. Disruption of ORF-RP3, located 190 bp upstream of bla OXA-60 and divergently transcribed, abolished induction of both β-lactamases. ORF-RP3, which encoded a polypeptide of 532 aa with an estimated molecular mass of 58.7 kDa, displayed no obvious sequence homology with known regulatory proteins. Trans-complementation of ORF-RP3 restored the basal and inducible expression of both oxacillinase genes, indicating that the induction of both enzymes was related to the presence of ORF-RP3. In addition to the loss of induction, inactivation of the ORF-RP3 in R. pickettii resulted in a complex pleiotropic phenotype, with increased lag phase and reduced survival after heat exposure, suggesting that ORF-RP3 might be a global regulator involved in unrelated regulatory pathways.
-
-
-
Identification of a diacylglycerol acyltransferase gene involved in accumulation of triacylglycerol in Mycobacterium tuberculosis under stress
Mycobacterium tuberculosis under stress stores triacylglycerol (TG). There are 15 genes in M. tuberculosis that belong to a novel family of TG synthase genes (tgs), but it is not known which of them is responsible for this accumulation of TG. In this paper, it is reported that M. tuberculosis H37Rv accumulated TG under acidic, static or hypoxic growth conditions, or upon treatment with NO, whereas TG accumulation was drastically reduced in the tgs1 (Rv3130c) disrupted mutant. Complementation with tgs1 restored this TG accumulation. C26 was a major fatty acid in this TG, indicating that the TGS1 gene product uses C26 fatty acid, which is known to be produced by the mycobacterial fatty acid synthase. TGS1 expressed in Escherichia coli preferred C26 : 0-CoA for TG synthesis. If TG storage is needed for the long-term survival of M. tuberculosis under dormant conditions, the tgs1 product could be a suitable target for antilatency drugs.
-
-
-
Identification of the Mycobacterium tuberculosis GlnE promoter and its response to nitrogen availability
More LessAdenylyltransferase, GlnE, has a predicted role in controlling the enzymic activity of glutamine synthetase, the key enzyme in ammonia assimilation. It was previously demonstrated that glnE is an essential gene in Mycobacterium tuberculosis. glnE is located downstream of glnA2, one of four glutamine synthetases. The expression of GlnE under various conditions was determined. Although a co-transcript of glnA2 and glnE was detectable, the major transcript was monocistronic. A transcriptional start site immediately upstream of glnE was identified and it was shown by site-directed mutagenesis that the predicted −10 region is a functional promoter. It was demonstrated that in a Mycobacterium smegmatis background M. tuberculosis PglnE was up-regulated in ammonia- or glutamine-containing media.
-
-
-
AopP, a type III effector protein of Aeromonas salmonicida, inhibits the NF-κB signalling pathway
Aeromonas salmonicida subsp. salmonicida contains a functional type III secretion system that is responsible for the secretion of the ADP-ribosylating toxin AexT. In this study, the authors identified AopP as a second effector protein secreted by this system. The aopP gene was detected in both typical and atypical A. salmonicida isolates and was found to be encoded on a small plasmid of approximately 6.4 kb. Sequence analysis indicates that AopP is a member of the YopJ family of effector proteins, a group of proteins that interfere with mitogen-activated protein kinase (MAPK) and/or nuclear factor kappa B (NF-κB) signalling pathways. AopP inhibits the NF-κB pathway downstream of IκB kinase (IKK) activation, while a catalytically inactivated mutant, AopPC177A, does not possess this inhibitory effect. Unlike other effectors of the YopJ family, such as YopJ and VopA, AopP does not inhibit the MAPK signalling pathway.
-
-
-
Mechanisms of copper loading on the Schizosaccharomyces pombe copper amine oxidase 1 expressed in Saccharomyces cerevisiae
More LessCopper amine oxidases (CAOs) are found in almost every living kingdom. Although Saccharomyces cerevisiae is one of the few yeast species that lacks an endogenous CAO, heterologous gene expression of CAOs from other organisms produces a functional enzyme. To begin to characterize their function and mechanisms of copper acquisition, two putative cao + genes from Schizosaccharomyces pombe were expressed in S. cerevisiae. Expression of spao1 + resulted in the production of an active enzyme capable of catalysing the oxidative deamination of primary amines. On the other hand, expression of spao2 + failed to produce an active CAO. Using a functional spao1 +–GFP fusion allele, the SPAO1 protein was localized in the cytosol. Under copper-limiting conditions, yeast cells harbouring deletions of the MAC1, CTR1 and CTR3 genes were defective in amine oxidase activity. Likewise, atx1Δ null cells exhibited no CAO activity, while ccc2Δ mutant cells exhibited decreased levels of amine oxidase activity, and mutations in cox17Δ and ccs1Δ did not cause any defects in this activity. Copper-deprived S. cerevisiae cells expressing spao1 + required a functional atx1 + gene for growth on minimal medium containing ethylamine as the sole nitrogen source. Under these conditions, the inability of the atx1Δ cells to utilize ethylamine correlated with the lack of SPAO1 activity, in spite of the efficient expression of the protein. Cells carrying a disrupted ccc2Δ allele exhibited only weak growth on ethylamine medium containing a copper chelator. The results of these studies reveal that expression of the heterologous spao1 + gene in S. cerevisiae is required for its growth in medium containing ethylamine as the sole nitrogen source, and that expression of an active Schiz. pombe SPAO1 protein in S. cerevisiae depends on the acquisition of copper through the high-affinity copper transporters Ctr1 and Ctr3, and the copper chaperone Atx1.
-
-
-
In vivo monitoring of the potassium channel KcsA in Streptomyces lividans hyphae using immuno-electron microscopy and energy-filtering transmission electron microscopy
More LessThe previous discovery of the Streptomyces lividans kcsA gene and its overexpression followed by the functional reconstitution of the purified gene product has resulted in new strategies to explore this channel protein in vitro. KcsA has evolved as a general model to investigate the structure/function relationship of ion channel proteins. Using specific antibodies raised against a domain of KcsA lacking membrane-spanning regions, KcsA has now been localized within numerous separated clusters between the outer face of the cytoplasm and the cell envelope in substrate hyphae of the S. lividans wild-type strain but not in a designed chromosomal disruption mutant ΔK, lacking a functional kcsA gene. Previous findings had revealed that caesium ions led to a block of KcsA channel activity within S. lividans protoplasts fused to giant vesicles. As caesium can be scored by electron energy loss spectroscopy better than potassium, this technique was applied to hyphae that had been briefly exposed to caesium instead of potassium ions. Caesium was found preferentially at the cell envelope. Compared to the ΔK mutant, the relative level of caesium was ≈30 % enhanced in the wild-type. This is attributed to the presence of KcsA channels. Additional visualization by electron spectroscopic imaging supported this conclusion. The data presented are believed to represent the first demonstration of in vivo monitoring of KcsA in its original host.
-
- Biodiversity And Evolution
-
-
-
DNA secretion and gene-level selection in bacteria
More LessNatural genetic transformation can facilitate gene transfer in many genera of bacteria and requires the presence of extracellular DNA. Although cell lysis can contribute to this extracellular DNA pool, several studies have suggested that the secretion of DNA from living bacteria may also provide genetic material for transformation. This paper reviews the evidence for specific secretion of DNA from intact bacteria into the extracellular environment and examines this behaviour from a population-genetics perspective. A mathematical model demonstrates that the joint action of DNA secretion and transformation creates a novel type of gene-level natural selection. This model demonstrates that gene-level selection could explain the existence of DNA secretion mechanisms that provide no benefit to individual cells or populations of bacteria. Additionally, the model predicts that any trait affecting DNA secretion will experience selection at the gene level in a transforming population. This analysis confirms that the secretion of DNA from intact bacterial cells is fully explicable with evolutionary theory, and reveals a novel mechanism for bacterial evolution.
-
-
-
-
Metabolic fingerprints of Mycobacterium bovis cluster with molecular type: implications for genotype–phenotype links
More LessMycobacterium bovis is the causative agent of bovine tuberculosis. Various genetic typing techniques have been used to trace the reservoirs of infection; however, they have limited success in population genetics and outbreak studies. Fourier-transform infrared spectroscopy (FT-IR) is a rapid phenotypic typing technique, which may be used to generate a metabolic fingerprinting and is increasingly used to characterize bacteria. When coupled with multivariate cluster analysis, this powerful combination has sufficient resolving power to discriminate bacteria down to subspecies level; however, to date this method has not been used in the differentiation of mycobacteria. Multiple isolates of the ten major spoligotypes in the UK, recovered from different geographical locations, were analysed using FT-IR. Hierarchical cluster analysis of the spectra showed that the isolates could be differentiated according to their spoligotypes. Six of the spoligotype FT-IR clusters were very homogeneous and all isolates were recovered together. However, the remaining four groups displayed a more heterogeneous phenotype, which may reflect greater variation than previously suspected within these groups. Included in the ten spoligotypes are the two most dominant isolates in the UK, designated types 9 and 17. Whilst type 17 showed a highly conserved phenotype as judged by FT-IR, type 9 showed a very heterogeneous metabolic profile and isolates were recovered throughout the dendrogram. This variation in type 9 is reflected in the high degree of diversity observed by variable number tandem repeats (VNTR) analysis, underlining the exquisite resolving power of FT-IR.
-
-
-
Biofilm formation by a Bacillus subtilis strain that produces γ-polyglutamate
The extracellular matrix produced by Bacillus subtilis B-1, an environmental strain that forms robust floating biofilms, was purified, and determined to be composed predominantly of γ-polyglutamate (γ-PGA), with a molecular mass of over 1000 kDa. Both biofilm formation and γ-PGA production by B. subtilis B-1 increased with increasing Mn2+ or glycerol concentration. γ-PGA was produced in a growth-associated manner in standing culture, and floating biofilms were formed. However, γ-PGA was produced in a non-growth-associated manner in shaking culture conditions. When B. subtilis B-1 was grown in a microaerated culture system, floating biofilm formation and γ-PGA production were significantly retarded, suggesting that oxygen depletion is involved in the initial steps of floating biofilm formation in standing culture. Proteomic analysis of membrane proteins demonstrated that flagellin, oligopeptide permease and Vpr protease precursor were the major proteins produced by cells in a floating biofilm and a colony.
-
- Environmental Microbiology
-
-
-
Bacterial factors influencing adhesion of Pseudomonas aeruginosa strains to a poly(ethylene oxide) brush
More LessMost bacterial strains adhere poorly to poly(ethylene oxide) (PEO)-brush coatings, with the exception of a Pseudomonas aeruginosa strain. The aim of this study was to find factors determining whether P. aeruginosa strains do or do not adhere to a PEO-brush coating in a parallel plate flow chamber. On the basis of their adhesion, a distinction could be made between three adhesive and three non-adhesive strains of P. aeruginosa, while bacterial motilities and zeta potentials were comparable for all six strains. However, water contact angles indicated that the adhesive strains were much more hydrophobic than the non-adhesive strains. Furthermore, only adhesive strains released surfactive extracellular substances, which may be engaged in attractive interactions with the PEO chains. Atomic force microscopy showed that the adhesion energy, measured from the retract curves of a bacterial-coated cantilever from a brush coating, was significantly more negative for adhesive strains than for non-adhesive strains (P<0.001). Through surface thermodynamic and extended-DLVO (Derjaguin, Landau, Verwey, Overbeek) analyses, these stronger adhesion energies could be attributed to acid–base interactions. However, the energies of adhesion of all strains to a brush coating were small when compared with their energies of adhesion to a glass surface. Accordingly, even the adhesive P. aeruginosa strains could be easily removed from a PEO-brush coating by the passage of a liquid–air interface. In conclusion, cell surface hydrophobicity and surfactant release are the main factors involved in adhesion of P. aeruginosa strains to PEO-brush coatings.
-
-
-
-
Plasmids from freshwater environments capable of IncQ retrotransfer are diverse and include pQKH54, a new IncP-1 subgroup archetype
Nine mercury-resistance plasmids isolated from river epilithon were assessed for their ability to retrotransfer the non-conjugative IncQ plasmid, R300B, derivatives of which have commercial uses that may result in accidental or deliberate release into the environment. Retrotransfer frequencies ranging from 2.1×10−4 to 1.75×10−5 were obtained for five of the nine plasmids – the remaining plasmids showed low or undetectable retrotransfer ability. The majority of the retrotransfer-proficient plasmids could not be classified by the tests used. Classical incompatibility testing with RP4 identified pQKH6, pQKH54 and pQM719 as IncP-1. Hybridization to replicon probes confirmed this for pQKH6 and pQM719 and added pQKH33. PCR with primers designed to amplify trfA and korA regions of IncP-1 plasmids did not identify any other plasmids. Plasmids pQKH6 and pQM719 but not pQKH54 produced similar SphI restriction profiles to the IncP-1β subgroup. The complete nucleotide sequence of pQKH54 was determined, revealing it to have a complete IncP-1 backbone but belonging to a new distinct subgroup which was designated IncP-1γ. The results emphasize the ubiquity and diversity of IncP-1 plasmids in the environment but demonstrate that plasmids of as yet unknown groups are also able to retrotransfer IncQ plasmids efficiently.
-
-
-
Anaerobic and aerobic metabolism of glycogen-accumulating organisms selected with propionate as the sole carbon source
More LessIn the microbial competition observed in enhanced biological phosphorus removal (EBPR) systems, an undesirable group of micro-organisms known as glycogen-accumulating organisms (GAOs) compete for carbon in the anaerobic period with the desired polyphosphate-accumulating organisms (PAOs). Some studies have suggested that a propionate carbon source provides PAOs with a competitive advantage over GAOs in EBPR systems; however, the metabolism of GAOs with this carbon source has not been previously investigated. In this study, GAOs were enriched in a laboratory-scale bioreactor with propionate as the sole carbon source, in an effort to better understand their biochemical processes. Based on comprehensive solid-, liquid- and gas-phase chemical analytical data from the bioreactor, a metabolic model was proposed for the metabolism of propionate by GAOs. The model adequately described the anaerobic stoichiometry observed through chemical analysis, and can be a valuable tool for further investigation of the competition between PAOs and GAOs, and for the optimization of the EBPR process. A group of Alphaproteobacteria dominated the biomass (96 % of Bacteria) from this bioreactor, while post-fluorescence in situ hybridization (FISH) chemical staining confirmed that these Alphaproteobacteria produced poly-β-hydroxyalkanoates (PHAs) anaerobically and utilized them aerobically, demonstrating that they were putative GAOs. Some of the Alphaproteobacteria were related to Defluvicoccus vanus (16 % of Bacteria), but the specific identity of many could not be determined by FISH. Further investigation into the identity of other GAOs is necessary.
-
- Genes And Genomes
-
-
-
Prediction of whole-genome DNA–DNA similarity, determination of G+C content and phylogenetic analysis within the family Pasteurellaceae by multilocus sequence analysis (MLSA)
More LessGenome predictions based on selected genes would be a very welcome approach for taxonomic studies, including DNA–DNA similarity, G+C content and representative phylogeny of bacteria. At present, DNA–DNA hybridizations are still considered the gold standard in species descriptions. However, this method is time-consuming and troublesome, and datasets can vary significantly between experiments as well as between laboratories. For the same reasons, full matrix hybridizations are rarely performed, weakening the significance of the results obtained. The authors established a universal sequencing approach for the three genes recN, rpoA and thdF for the Pasteurellaceae, and determined if the sequences could be used for predicting DNA–DNA relatedness within the family. The sequence-based similarity values calculated using a previously published formula proved most useful for species and genus separation, indicating that this method provides better resolution and no experimental variation compared to hybridization. By this method, cross-comparisons within the family over species and genus borders easily become possible. The three genes also serve as an indicator of the genome G+C content of a species. A mean divergence of around 1 % was observed from the classical method, which in itself has poor reproducibility. Finally, the three genes can be used alone or in combination with already-established 16S rRNA, rpoB and infB gene-sequencing strategies in a multisequence-based phylogeny for the family Pasteurellaceae. It is proposed to use the three sequences as a taxonomic tool, replacing DNA–DNA hybridization.
-
-
-
-
EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. I. Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia
More LessThe entomopathogenic fungus Beauveria (Cordyceps) bassiana holds much promise as a pest biological control agent. B. bassiana produces at least three in vitro single cell infectious propagules, including aerial conidia, vegetative cells termed blastospores and submerged conidia, that display different morphological, biochemical and virulence properties. Populations of aerial conidia, blastospores and submerged conidia were produced on agar plates, rich liquid broth cultures and under conditions of nutrient limitation in submerged cultures, respectively. cDNA libraries were generated from mRNA isolated from each B. bassiana cell type and ∼2500 5′ end sequences were determined from each library. Sequences derived from aerial conidia clustered into 284 contigs and 963 singlets, with those derived from blastospores and submerged conidia forming 327 contigs with 788 singlets, and 303 contigs and 1079 contigs, respectively. Almost half (40–45 %) of the sequences in each library displayed either no significant similarity (e value >10−4) or similarity to hypothetical proteins found in the NCBI database. The expressed sequence tag dataset also included sequences representing a significant portion of proteins in cellular metabolism, information storage and processing, transport and cell processes, including cell division and posttranslational modifications. Transcripts encoding a diverse array of pathogenicity-related genes, including proteases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites, were also identified. Comparative analysis between the libraries identified 2416 unique sequences, of which 20–30 % were unique to each library, and only ∼6 % of the sequences were shared between all three libraries. The unique and divergent representation of the B. bassiana transcriptome in the cDNA libraries from each cell type suggests robust differential gene expression profiles in response to environmental conditions.
-
-
-
EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. II. Fungal cells sporulating on chitin and producing oosporein
More LessIn the accompanying paper [ Cho, E.-M., Liu, L., Farmerie, W. & Keyhani, N. O. (2006) . Microbiology 152, 2843–2854], the analysis of expressed sequence tag (EST) libraries derived from homogeneous single-cell populations of aerial conidia, in vitro blastospores and submerged conidia of the entomopathogenic fungus Beauveria (Cordyceps) bassiana has been reported. Here an extended EST analysis is presented of complex cell mixtures derived from fungal cells sporulating on chitin or grown under culture conditions inducing the production of the B. bassiana secondary metabolite, oosporein. Fungal material used for the construction of the libraries included germinating conidia and blastospores, mycelia, as well as cells in various developmental stages. Approximately 2500 5′ end sequences were determined from random sequencing of clones from each library, and were clustered into 277 contigs with 1069 singlets, and 306 contigs with 1064 singlets, for the chitin and oosporein libraries, respectively. Almost half (45–50 %) of the sequences in each library displayed either no significant similarity (e value >10−4) or similarity to hypothetical proteins found in the NCBI database. Approximately 20–25 % of the sequences in each library could be annotated by gene ontology terms. A comparative analysis between the two libraries, as well as the libraries in the accompanying paper, is presented. A set of 4360 clustered and unique sequences was characterized. The data are indicative of a highly plastic gene expression repertoire being available to B. bassiana for growth during different environmental and developmental conditions, and provides a dataset for gene discovery and genome annotation.
-
- Pathogens And Pathogenicity
-
-
-
Identification and characterization of msa (SA1233), a gene involved in expression of SarA and several virulence factors in Staphylococcus aureus
More LessThe staphylococcal accessory regulator (sarA) plays a central role in the regulation of virulence in Staphylococcus aureus. To date, studies involving sarA have focused on its activity as a global regulator that modulates transcription of a wide variety of genes (>100) and its role in virulence. However, there is also evidence to suggest the existence of accessory elements that modulate SarA production and/or function. A reporter system was developed to identify such elements, and a new gene, msa (SA1233), mutation of which results in reduced expression of SarA, was identified and characterized. Additionally, it was shown that mutation of msa resulted in altered transcription of the accessory gene regulator (agr) and the genes encoding several virulence factors including alpha toxin (hla) and protein A (spa). However, the impact of mutating msa was different in the laboratory strain RN6390 and the clinical isolate UAMS-1. For instance, mutation of msa caused a decrease in spa and hla transcription in RN6390 but had a different effect in UAMS-1. The strain-dependent effects of the msa mutation were similar to those observed previously, which suggests that msa may modulate the production of specific virulence factors through its impact on sarA. Interestingly, sequence analysis of Msa suggests that it is a putative membrane protein with three membrane-spanning regions, indicating that Msa might interact with the environment. The findings show that msa is involved in the expression of SarA and several virulence factors.
-
-
-
-
Implication of (Mn)superoxide dismutase of Enterococcus faecalis in oxidative stress responses and survival inside macrophages
The gene encoding the manganese-containing superoxide dismutase (MnSOD) of Enterococcus faecalis was characterized. It is transcribed monocistronically from an upstream promoter identified by rapid amplification of cDNA ends (RACE)-PCR. A sodA mutant was constructed and characterized. Growth of the mutant strain was not significantly different from that of its wild-type counterpart in standing and aerated cultures. However, the mutant was more sensitive towards menadione and hydroperoxide stresses. The response to H2O2 stress was analysed in more detail, and the mode of killing of this oxidant was different under anaerobic and aerobic conditions. Cultures grown and challenged under anaerobic conditions were highly sensitive to treatment with 35 mM H2O2. They were largely protected by the iron chelator deferoxamine, which suggested that killing was mainly due to an enhanced Fenton reaction. In contrast, neither strain was protected by the iron chelators deferoxamine and diethylenetriaminepentaacteic acid when grown and challenged under aerobic conditions, which suggested that inactivation of the cells by H2O2 was due to another killing mode. The sodA mutant was more sensitive under these conditions, showing that MnSOD is also important for protecting the cells from damage under aerobic conditions. Finally, the MnSOD of Ent. faecalis may be considered to be a virulence factor, since survival of the corresponding mutant strain was highly affected inside mouse peritoneal macrophages.
-
-
-
Irr regulates brucebactin and 2,3-dihydroxybenzoic acid biosynthesis, and is implicated in the oxidative stress resistance and intracellular survival of Brucella abortus
More LessBrucella abortus faces iron deprivation in both nature and the host. To overcome this limitation, Brucella secretes the siderophores 2,3-dihydroxybenzoic acid and brucebactin. A Fur-like protein named Irr has previously been characterized in B. abortus; this protein is present in the α-2 group of Proteobacteria only, where it negatively regulates haem biosynthesis when iron is scarce. Additional evidence that Irr also regulates the synthesis of both siderophores is presented here. Transcriptional lacZ fusion and chemical determinations revealed that Irr induced the transcription of the operon involved in the synthesis of the catecholic siderophores, which were consequently secreted under conditions of iron limitation. Irr was able to bind the upstream region of the operon, as shown by electrophoretic mobility shift assay. A B. abortus irr mutant showed higher intracellular haem content, catalase activity and resistance to hydrogen peroxide than the wild-type strain. The mutation also improved the replication and survival of iron-depleted bacteria within cultured mammalian cells. Although the pathogenesis of Brucella correlates with its ability to replicate intracellularly, pathogenicity was not attenuated when assayed in a murine model.
-
-
-
Transcriptional profiling of Borrelia burgdorferi containing a unique bosR allele identifies a putative oxidative stress regulon
More LessBorrelia burgdorferi regulates gene expression in response to environmental conditions, including temperature, pH, redox potential and host factors. B. burgdorferi encodes a PerR homologue designated BosR, which presumably serves as a global regulator of genes involved in the oxidative stress response. Infectious B. burgdorferi strain B31 is resistant to oxidative stressors in vitro, whereas the non-infectious isolate was sensitive due, in part, to a point mutation that converts an arginine to a lysine at residue 39 of BosR. Subsequent insertional inactivation of this bosRR39K allele (bosRR39K : : kanR) restored resistance to oxidative stressors. These observations suggest that the B. burgdorferi non-infectious bosRR39K : : kanR strain may transcribe genes that are also expressed in infectious B. burgdorferi cells, but are repressed in the bosRR39K background, thus explaining the different oxidative stress phenotypes observed between these isolates. To test this hypothesis, macroarray technology and quantitative RT-PCR were utilized to compare the transcriptional profiles from the isogenic bosRR39K and bosRR39K : : kanR isolates. Array data indicated that 88 ORFs were significantly expressed in the absence of BosRR39K. Since most affected genes mapped to the chromosome, it is likely that these genes define an important physiologic response for B. burgdorferi. Included within the genes identified was the detoxification gene sodA, as well as other loci not overtly linked to oxidative stress. These results suggest that a putative BosR regulon, as defined by the bosRR39K allele, is required to combat toxic oxidative intermediates, but may also be involved in adaptive strategies that are independent of reactive oxygen species.
-
-
-
Mycobacterium smegmatis whmD and its homologue Mycobacterium tuberculosis whiB2 are functionally equivalent
More LessMycobacterium smegmatis whmD is is an essential gene involved in cell division. This paper shows that whmD and its homologue whiB2 in Mycobacterium tuberculosis are functionally equivalent. The genes are syntenous, and share significant homology in both their coding and non-coding DNA sequences. Transcription site mapping showed that the two genes possess near-identical promoter elements, and they displayed comparable promoter strengths in a reporter gene assay. The two proteins show near identity in their C-terminus, and polyclonal antiserum to WhmD specifically cross-reacts with a ∼15 kDa band in M. tuberculosis lysates. Following overexpression of sense and anti-sense constructs in their cognate mycobacterial hosts, whiB2 and whmD transformants displayed a small-colony phenotype, exhibited filamentation, and showed a reduction in viability. These observations reveal that the two proteins are functionally homologous and that their intracellular concentration is critical for septation in mycobacteria. Colonies of M. tuberculosis overexpressing whiB2 were spherical and glossy, suggesting a change in composition of the cell envelope. Filaments of the conditionally complemented M. smegmatis whmD mutant were non-acid-fast, also indicating changes in characteristics of surface lipids. M. smegmatis transformants carrying a whmD–gfp fusion showed a diffuse pattern of fluorescence, consistent with the putative role of WhmD as a regulator. These observations strongly suggest that M. tuberculosis whiB2 is an essential gene and its protein product in all likelihood regulates the expression of genes involved in the cell division cascade.
-
-
-
Regulation of the expression of whiB1 in Mycobacterium tuberculosis: role of cAMP receptor protein
More LessThe wbl (whiB-like) genes encode putative transcription factors unique to actinomycetes. This study characterized the promoter element of one of the seven wbl genes of Mycobacterium tuberculosis, whiB1 (Rv3219c). The results reveal that whiB1 is transcribed by a class I-type cAMP receptor protein (CRP)-dependent promoter, harbouring a CRP-binding site positioned at −58.5 with respect to its transcription start point. In vivo promoter activity analysis and electrophoretic mobility shift assays suggest that the expression of whiB1 is indeed regulated by cAMP-dependent binding of CRPM (encoded by the M. tuberculosis gene Rv3676) to the whiB1 5′ untranslated region (5′UTR). β-Galactosidase gene fusion analysis revealed induction of the whiB1 promoter in M. tuberculosis on addition of exogenous dibutyric cAMP (a diffusible cAMP analogue) only when an intact CRP-binding site was present. These results indicate that M. tuberculosis whiB1 transcription is regulated in part by cAMP levels via direct binding of cAMP-activated CRPM to a consensus CRP-binding site in the whiB1 5′UTR.
-
-
-
Laboratory and clinical Pseudomonas aeruginosa strains do not bind glycosphingolipids in vitro or during type IV pili-mediated initial host cell attachment
The glycosphingolipids (GSLs) gangliotriaosylceramide (Gg3) and gangliotetraosylceramide (Gg4) have been implicated as receptors for type IV pili (T4P)-mediated Pseudomonas aeruginosa epithelial cell attachment. Since P. aeruginosa T4P are divided into five groups, the authors determined whether GSLs in general, and Gg3 and Gg4 in particular, are specifically bound and required for host epithelial cell attachment of clinical and laboratory strains within these groups. An enterohaemorrhagic Escherichia coli strain, CL56, known to bind to both Gg3 and Gg4, provided a positive control. TLC overlay showed no binding of more than 12 P. aeruginosa strains to either Gg3 or Gg4 (or other GSLs), while CL56 Gg3/Gg4 binding was readily detectable. GSL ELISA similarly demonstrated no significant P. aeruginosa binding to Gg3 or Gg4, compared with CL56. Using a selective chemical inhibitor, epithelial cell GSL synthesis was abrogated, and Gg3 and Gg4 expression deleted, but P. aeruginosa attachment was not impaired. Target cell attachment was mediated by T4P, since non-piliated, but flagellated, mutants were unable to bind to the target cells. CFTR (cystic fibrosis transmembrane conductance regulator) has also been implicated as a receptor; however, in this work, overexpression of CFTR had no effect on P. aeruginosa binding. It is concluded that neither Gg3 nor Gg4 are specifically recognized by P. aeruginosa, and that endogenous GSLs do not have a role in the attachment of live intact P. aeruginosa to cultured lung epithelial cells. In contrast to whole piliated P. aeruginosa, T4P sheared from such bacteria showed significant Gg3 and Gg4 binding, which may explain the results of other studies.
-
- Physiology
-
-
-
Construction and characterization of a Lactococcus lactis strain deficient in intracellular ClpP and extracellular HtrA proteases
A Lactococcus lactis strain deficient in both its major proteases, intracellular (ClpP) and extracellular (HtrA), was constructed and characterized. This strain, hereafter called clpP-htrA, could be obtained only by conjugation between a clpP donor strain and an htrA recipient strain in the NZ9000 context, allowing heterologous gene expression under the control of the NICE (nisin-controlled expression) system. The clpP-htrA double mutant showed both higher stress tolerance (e.g. high temperature and ethanol resistance) and higher viability than single clpP or htrA mutant strains. In addition, the secretion rate of two heterologous proteins (staphylococcal nuclease Nuc and Nuc-E7) was also higher in clpP-htrA than in the wild-type strain. This strain should be a useful host for high-level production and quality of stable heterologous proteins.
-
-
-
-
Fermentation acids inhibit amino acid deamination by Clostridium sporogenes MD1 via a mechanism involving a decline in intracellular glutamate rather than protonmotive force
More LessFermentation acids inhibited the growth and ammonia production of the amino-acid-fermenting bacterium Clostridium sporogenes MD1, but only when the pH was acidic. Such inhibition was traditionally explained by the ability of fermentation acids to act as uncouplers and decrease protonmotive force (Δp), but C. sporogenes MD1 grows even if the Δp is very low. Cell suspensions incubated with additional sodium chloride produced ammonia as rapidly at pH 5.0 as at pH 7.0, but cells incubated with additional sodium lactate were sensitive to even small decreases in extracellular pH. Similar results were obtained if the sodium lactate was replaced by sodium acetate or propionate. When extracellular pH declined, ΔpH increased even if sodium lactate was present. The cells accumulated intracellular lactate anion when the pH was acidic, and intracellular glutamate declined. Because amino acid deamination is linked to a transamination reaction involving glutamate dehydrogenase, the decrease in ammonia production could be explained by the decrease in intracellular glutamate. This latter hypothesis was consistent with the observation that extracellular glutamate addition restored amino acid deamination even though glutamate alone did not allow for the generation of ammonia.
-
-
-
Trehalose metabolism is important for heat stress tolerance and spore germination of Botrytis cinerea
More LessTo analyse the role of trehalose as stress protectant and carbon storage compound in the grey mould fungus Botrytis cinerea, mutants defective in trehalose-6-phosphate synthase (TPS1) and neutral trehalase (TRE1) were constructed. The Δtps1 mutant was unable to synthesize trehalose, whereas the Δtre1 mutant showed elevated trehalose levels compared to the wild-type and was unable to mobilize trehalose during conidial germination. Both mutants showed normal vegetative growth and were not affected in plant pathogenicity. Growth of the Δtps1 mutant was more heat sensitive compared to the wild-type. Similarly, Δtps1 conidia showed a shorter survival under heat stress, and their viability at moderate temperatures was strongly reduced. In germinating wild-type conidia, rapid trehalose degradation occurred only when germination was induced in the presence of nutrients. In contrast, little trehalose breakdown was observed during germination on hydrophobic surfaces in water. Here, addition of cAMP to conidia induced trehalose mobilization and accelerated the germination process, probably by activation of TRE1. In accordance with these data, both mutants showed germination defects only in the presence of sugars but not on hydrophobic surfaces in the absence of nutrients. The data indicate that in B. cinerea trehalose serves as a stress protectant, and also as a significant but not essential carbon source for germination when external nutrients are low. In addition, evidence was obtained that trehalose 6-phosphate plays a role as a regulator of glycolysis during germination.
-
-
-
Involvement of the S-layer proteins Hpi and SlpA in the maintenance of cell envelope integrity in Deinococcus radiodurans R1
More LessThe potential functions have been investigated of two proteins in Deinococcus radiodurans R1 predicted to be involved in the maintenance and integrity of the S layer: the hexagonally packed intermediate (Hpi) protein, and SlpA (DR2577), a homologue of an S-layer SlpA protein in Thermus thermophilus. Deletion of the hpi gene had little effect on the structure of the cell envelope or on shear- or solvent-induced stress responses. However, deletion of the slpA gene caused substantial alterations in cell envelope structure, and a significant defect in resistance to solvent and shear stresses compared to the wild-type. Ultrastructural analysis of slpA mutant cells indicated loss of much of the outer Hpi protein carbohydrate coat, the ‘pink envelope’, and the membrane-like backing layer. Together these results suggest that the SlpA protein may be involved in attachment of the Hpi surface layer to the inner cell envelope, and that SlpA may play an important role in the maintenance of cell envelope integrity in D. radiodurans.
-
- Plant-Microbe Interactions
-
-
-
Plasmid pSci6 from Spiroplasma citri GII-3 confers insect transmissibility to the non-transmissible strain S. citri 44
More LessThe insect-transmissible strain GII-3 of Spiroplasma citri contains plasmids pSci1–6, five of which (pSci1–5) encode adhesin-like proteins and one (pSci6) encodes protein P32, which has been associated with insect transmissibility. In contrast, S. citri strains ASP-1 and 44, which cannot be transmitted via injection into the leafhopper vector Circulifer haematoceps, lack these proteins and also do not carry plasmids pSci1–6. To further study the apparent relationship between the presence of plasmids and insect transmissibility, plasmids from S. citri GII-3 were introduced into the insect-non-transmissible S. citri strain 44 by electrotransformation using the tetM gene as the selection marker. Tetracycline-resistant transformants were shown to carry one, two or three distinct plasmids. Plasmids pSci1–6 were all detected in the transformants, pSci1 being the most frequently found, alone or together with other plasmids. Selected S. citri 44 transformants having distinct plasmid contents were submitted, separately or in combination, to experimental transmission to periwinkle (Catharanthus roseus) plants via injection into the leafhopper vector. The occurrence of symptomatic plants indicated that, in contrast to S. citri 44, spiroplasmal transformants were transmitted to the host plant, in which they multiplied. Spiroplasma cultures isolated from these infected plants all contained pSci6, leading to the conclusion that, under the experimental conditions used, transformation by pSci6 conferred insect transmissibility to S. citri strain 44. This is believed to be the first report of a phenotypic change associated with transformation of S. citri by natural plasmids.
-
-
- Corrigendum
-
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)
Most Read This Month
![Loading](/images/jp/spinner.gif)