- Volume 144, Issue 8, 1998
Volume 144, Issue 8, 1998
- Review Article
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- Microbiology Comment
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- Biochemistry
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Conservation of the amino-terminal epitope of elongation factor Tu in eubacteria and archaea
More LessAn epitope of elongation factor Tu (EF-Tu), which is found in organisms in both the bacterial and archaeal domains, was recently defined by mAb 900. To localize the conserved epitope within the EF-Tu molecule and to determine its sequence, SPOTScan analysis of synthetic peptides, Western blot analysis of purified EF-Tu domains and site-directed mutagenesis studies were used. Analysis of mAb 900 binding to overlapping 15-mer peptides encompassing the complete sequence of EF-Tu of Escherichia coli was inconclusive, suggesting three distinct regions may be epitopes. Western blot analysis of EF-Tu domains 1-3 of Thermus thermophilus suggested that the epitope was located at the N terminus. This was confirmed by site-directed mutagenesis of EF-Tu domain 1 of Mycoplasma hominis. By C-terminal truncation of the N-terminal 15-mer peptide the epitope was mapped to EF-Tu residues 1-6. Replacement of each of the residues in the epitope peptide demonstrated that only positions 5 and 6 were indispensable for antibody binding. These data provide evidence that the highly conserved epitope recognized by mAb 900 in the bacterial and archaeal domains is located at the very end of the N terminus of the EF-Tu molecule.
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- Bioenergetics And Transport
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Redox poise and oxygenation of cytochrome bd in the diazotroph Azotobacter vinelandii assessed in vivo using diode-array reflectance spectrophotometry
More LessA ferrous oxygenated form of cytochrome d is characteristic of all cytochrome bd-type oxidases so far examined, but its participation in enzyme turnover is unclear. It is relatively stable, occurs in aerated cell suspensions and predominates during enzyme preparation. In this study, diode-array reflectance spectrophotometry was used to assess the redox poise and oxygenation of cytochrome bd in vivo, in the aerobic diazotroph Azotobacter vinelandii. Mutants either lacking or overproducing the cytochrome bd oxidase were used to confirm the reliability of the optical configuration. Changes in absorbance attributed to cytochromes b, c and d were followed as the O2 supply was altered either in suspensions of harvested cells or during steady-state growth. In washed cell suspensions, three states of cytochrome d, which differed in absorbance characteristics, were seen: (1) an oxygenated form that absorbs at 650 nm, (2) a form which has little absorbance at either 650 or 630 nm and (3) the reduced form that absorbs at 630 nm. The transition between states 2 and 3, but not 1 and 2, correlated with the changes in the redox states of cytochromes b 595 and b 560. The dissolved O2 concentration at which this transition occurred coincided approximately with the apparent O2 affinity for the oxidase in vivo (approx. 5 M). During steady-state growth, the cytochromes were partially reduced and the oxygenated form of cytochrome d was undetected. These in situ measurements support the view that an oxygenated form of cytochrome d (absorbing at 650 nm) in the one-electron-reduced cytochrome bd-type oxidase does not take part in enzyme turnover.
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Cloning of the trkAH gene cluster and characterization of the Trk K+-uptake system of Vibrio alginolyticus
More LessK+-uptake genes of Vibrio alginolyticus were identified by cloning chromosomal DNA fragments of this organism into plasmids, followed by electroporation and selection for growth at low K+ concentrations of cells of an Escherichia coli strain defective in K+ uptake. A 4.1 kb DNA fragment contained a cluster of three ORFs on the same DNA strand: the previously identified trkA gene, a gene similar to E. coli trkH (V. alginolyticus trkH) and a new gene, orf1, whose function is not clear. Products of V. alginolyticus trkA and orf1 were detected in E. coli minicells. trkA and trkH from V. alginolyticus restored growth at low K+ concentrations of an E. coli ΔtrkA and an E. coli ΔtrkG ΔtrkH strain, respectively, suggesting that these V. alginolyticus genes can functionally replace their E. coli counterparts. In addition, a plasmid containing V. alginolyticus trkAH permitted growth of an E. coli ΔsapABCDF (ΔtrkE) strain at low K+ concentrations. This effect was mainly due to V. alginolyticus trkH and was enhanced by trkA from this organism. Measurements of net K+-uptake rates indicated that the presence of these genes in E. coli renders the Trk systems independent of products from the E. coli sapABCDF (trkE) operon.
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- Biotechnology
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Asymmetric reduction of racemic sulfoxides by dimethyl sulfoxide reductases from Rhodobacter capsulatus, Escherichia coli and Proteus species
The enantioselective reduction of racemic sulfoxides by dimethyl sulfoxide reductases from Rhodobacter capsulatus, Escherichia coli, Proteus mirabilis and Proteus vulgaris was investigated. Purified dimethyl sulfoxide reductase from Rhodobacter capsulatus catalysed the selective removal of (S)-methyl p-tolyl sulfoxide from a racemic mixture of methyl p-tolyl sulfoxide and resulted in an 88% recovery of enantiomerically pure (R)-methyl p-tolyl sulfoxide. Rhodobacter capsulatus was shown to be able to grow photoheterotrophically in the presence of certain chiral sulfoxides under conditions where a sulfoxide is needed as an electron sink. Whole cells of Rhodobacter capsulatus were shown to catalyse the enantioselective reduction of methyl p-tolyl sulfoxide, ethyl 2-pyridyl sulfoxide, methylthiomethyl methyl sulfoxide and methoxymethyl phenyl sulfoxide. Similarly, whole cells of Escherichia coli, Proteus mirabilis and Proteus vulgaris reduced these sulfoxides but with opposite enantioselectivity.
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- Development And Structure
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Cytochalasin D interferes with contractile actin ring and septum formation in Schizosaccharomyces japonicus var. versatilis
More LessThe cells of Schizosaccharomyces japonicus var. versatilis responded to the presence of cytochalasin D (CD), an inhibitor of actin polymerization, by the disappearance of contractile actin rings (ARs) that had already formed and by inhibition of new ring formation. Actin cables disappeared. Actin patches remained preserved and became co-localized with regions of actual cell wall formation (at cell poles and at the site of septum development). Removal of the AR arrested formation of the primary septum and led to the production of aberrant septum protrusions in that region. Nuclear division was accomplished in the presence of CD but new ARs were not produced. The wall (septum) material was deposited in the form of a wide band at the inner surface of the lateral cell wall in the cell centre. This layer showed a thin fibrillar structure. The removal of CD resulted in rapid formation of new ARs in the equatorial region of the cells. This implies that the signal for AR localization was not abolished either by CD effects or by removal of an AR already formed. Some of the newly developed ARs showed atypical localization and orientation. In addition, redundant, subcortically situated actin bundles were produced. The removal of CD was quickly followed by the development of primary septa co-localized with ARs. Wall protrusions occurred co-localized with the redundant actin bundles. If these were completed in a circle, redundant septa developed. The AR is a mechanism which, in time and space, triggers cytokinesis by building a septum sequentially dependent on the AR. Aberrant septa were not capable of separating daughter cells. However, non-separated daughter cells subsequently gave rise to normal cells.
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A hydrophobin (ABH3) specifically secreted by vegetatively growing hyphae of Agaricus bisporus (common white button mushroom)
More LessAerial mycelium and hyphal strands of Agaricus bisporus, strain U1, exhibited a rodlet pattern at their surfaces characteristic for assembled class I hydrophobins. An SDS-insoluble/trifluoroacetic-acid-soluble fraction from strands was found to contain one abundant protein with an apparent molecular mass on gel of 19 kDa. Two sequences for this protein (ABH3), typical of class I hydrophobins, could be deduced by sequencing cDNA clones obtained by RT-PCR. The two forms of the protein could be assigned to different alleles present in the two homokaryons that constitute the heterokaryotic U1 strain. ABH3 displays all the in vitro properties of a typical class I hydrophobin such as SC3 from Schizophyllum commune but is not glycosylated or otherwise post-translationally modified because the molecular mass values deduced from the amino acid sequence (9228 and 9271 Da) and derived from mass spectrometry were in good agreement. The ABH3 transcript was found to be present in the vegetative mycelium of both primary and secondary mycelium but not in the fruiting bodies, whereas the reverse was found for the ABH1 hydrophobin. Using an S. commune mutant with a disrupted SC3 gene it was found that ABH3 can substitute for SC3 in inducing formation of aerial hyphae, suggesting a role of ABH3 in the emergence of aerial hyphae and strands in A. bisporus.
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- Environmental Microbiology
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rRNA-targeted fluorescent in situ hybridization analysis of bacterial community structure in river water
More LessAn improved in situ hybridization technique, HNPP-FISH, using 2-hydroxy-3-naphthoic acid 2′-phenylanilide phosphate (HNPP) and Fast Red TR was applied to analyse the community structure of planktonic bacteria in river water. Oligonucleotide probes specific for the domain Bacteria (EUB338) and five bacterial groups [Flavobacterium-Cytophaga; Burkholderia-Pseudomonas (rRNA III)-authentic Alcaligenes; Vibrio-Aeromonas; Pseudomonas (rRNA I); the genus Acinetobacter] were used to investigate the bacterial community structure at two sites differing in organic carbon pollution level. At the eutrophic site, 54-68% of all cells visualized by staining with DAPI (4′,6-diamidino-2-phenylindole) could be detected with probe EUB338. In samples from the oligotrophic site, 39-45% of the total cells hybridized with EUB338. At the eutrophic site, approximately 50% of the total cells were identified with the five group-specific probes; the bacterial community structure was dominated by the Flavobacterium-Cytophaga group and Burkholderia-Pseudomonas (rRNA III)-authentic Alcaligenes group. At the oligotrophic site, only 26-38% of the total cells were identified with the five group-specific probes. The community structure at the oligotrophic site was similar to that at the eutrophic site, although the percentage of EUB338-detectable cells differed. No appreciable change was found in the community structure during the sampling period at either site. The improved HNPP-FISH technique should be a useful tool for the analysis of microbial community composition.
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Involvement of chitinases of Bacillus thuringiensis during pathogenesis in insects
More LessBacillus thuringiensis subsp. israelensis IPS78 and B. thuringiensis subsp. aizawai HD133 both secreted exochitinase activity when grown in a medium containing chitin. Allosamidin, a specific chitinase inhibitor, inhibited activity from both strains, with IC50 values of about 50 μM with colloidal chitin as substrate and between 1 and 10 μM with 4-methylumbelliferyl-diacetylchitobioside and 4-methylumbelliferyl-triacetylchitotrioside as substrates. The involvement of these chitinolytic activities during pathogenesis in insects has been investigated with B. thuringiensis subsp. israelensis IPS78 against larvae of the midge Culicoides nubeculosus, and with B. thuringiensis subsp. aizawai HD133 against caterpillars of the cotton leafworm Spodoptera littoralis. Presence of 100 μM allosamidin increased the LD50 by factors of 1.3 and 1.4, respectively, demonstrating a role for bacterial chitinases in the attack on the insects. Presence of chitinase A from Serratia marcescens considerably decreased the values for LD50' confirming previous observations with different systems of the potentiation of entomopathogenesis of B. thuringiensis by exogenous chitinases. The most likely action of the endogenous chitinases of B. thuringiensis is to weaken the insects' peritrophic membranes, allowing more ready access of the bacterial toxins to the gut epithelia. Addition of exogenous chitinases will then increase this effect. Complementary cross-infection experiments, strain HD133 against midge larvae and strain IPS78 against caterpillars, were performed to investigate the pathogen/host specificities of the effects. Results showed that much higher concentrations of bacteria were required to achieve even low mortalities, and addition of chitinase A gave no increase in death rate.
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Temporal changes in the frequency of colicinogeny in Escherichia coli from house mice
More LessEscherichia coli was isolated from feral house mice (Mus domesticus) during the course of a mouse plague in the state of Victoria, Australia. The isolates were characterized for the production of colicins and their resistance to the co-occurring colicins. Of the 447 isolates examined, 59% were found to be colicinogenic. Phenotypic and PCR-based genotypic methods were used to determine the types of colicins being produced. Colicin E2 was the most common, representing 27% of the colicin-producing isolates. Colicin la was produced by 3% of the colicinogenic isolates. The remaining colicins could not be identified, but phenotypic and PCR data argue that at least nine different colicin types are present in this collection of E. coli. The frequency of colicinogenic isolates declined from 71% to 43% over the 7 months of the study. All colicin types appeared to decline in frequency. Concurrently, the resistance of isolates to colicin E2 increased from about 50% to 70%. Two hypotheses are proposed to explain the decline in the frequency of colicinogeny in this population of E. coli. The first relates to the within-host interactions occurring among colicinogenic, colicin-susceptible and colicin-resistant populations within a host. The second relates to the among-host interactions between susceptible and colicinogenic populations and the effect of host population densities on these interactions.
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Manganous ions suppress photosynthesis gene expression in Rhodobacter sphaeroides
More LessThe effect of manganous ions [Mn(II)] and ferrous ions [Fe(II)] on expression of photosynthesis genes in Rhodobacter sphaeroides was investigated. The presence of Mn(II) during phototrophic (anaerobic) and chemotrophic (aerobic) growth of R. sphaeroides caused a decrease in the amount of bacteriochlorophyll and carotenoid pigments which were synthesized and this was associated mainly with a decrease in the level of light-harvesting complex II. Mn(II) was shown to cause a decrease in expression of the puc operon, which encodes the polypeptides of light-harvesting complex II. Expression of the puc operon is controlled by the central repressor of photosynthesis gene expression, PpsR. In a ppsR mutant there was no effect of Mn(II) on photosynthesis gene expression. It is concluded that Mn(II) may act as a compressor in the action of PpsR or act via an as yet uncharacterized protein that interacts with PpsR. In contrast to the effects of Mn(II), Fe(II) was required for high levels of photosynthesis gene expression. This requirement for Fe(II) was shown to be related to the regulation of hemA, a gene under the control of the transcriptional regulator, FnrL. Mn(II) did not affect FnrL-dependent gene expression.
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- Genetics And Molecular Biology
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The lipopolysaccharide biosynthesis locus of Campylobacter jejuni 81116
Most Campylobacter jejuni strains express lipo-oligosaccharides. Some strains also express lipopolysaccharides (LPS), with O-antigen-like carbohydrate repeats. C. jejuni 81116 expresses an LPS containing both lipo-oligosaccharides and O-antigen-like repeats, but nothing is known about the structure or sugar composition of these LPS species. A cosmid library of the genome of C. jejuni 81116 was constructed and probed with Campylobacter hyoilei genes involved in LPS synthesis. Five cosmids hybridized with the probe and two of these expressed C. jejuni 81116 LPS in Escherichia coli. By subcloning, a 16 kb DNA region was identified which contains the genetic information required to express C. jejuni LPS. DNA sequence analysis revealed 11 ORFs homologous to genes involved in LPS synthesis of other bacteria. They consisted of three homologues of sugar biosynthesis genes, two homologues of transport genes and six homologues of sugar transferases.
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Transcription of the trp operon in Lactococcus lactis is controlled by antitermination in the leader region
More LessThe regulatory functions of the leader region preceding the Lactococcus lactis trp operon have been studied by mutagenesis analysis. This leader presents striking similarity to ‘T-box’ leaders found upstream of many Gram-positive aminoacyl-tRNA synthetase genes and some amino acid biosynthesis operons, which are controlled by antitermination through interaction of the leader transcript with cognate uncharged tRNA. A region of the L. lactis leader transcript also contains a series of (G/U)AG repeats which, in Bacillus, are involved in the binding of the trp RNA-binding protein (TRAP) which controls trp transcription. A screen was developed for the isolation of regulatory mutants affected in the leader region. All spontaneous mutants contained deletions; point mutations were only obtained after UV-induced mutagenesis. All mutations affected the putative transcription terminator upstream of the trp operon, demonstrating that trp is indeed controlled by transcription antitermination.
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Transcription and transcript processing in the sdh CDAB-sucABCD operon of Escherichia coli
More LessThe genes encoding succinate dehydrogenase (sdhCDAB), the specific components of the 2-oxoglutarate dehydrogenase complex (ODH, E1o and E2o; sucAB) and succinyl-CoA synthetase (sucCD) form a cluster containing two promoters at 16 · 3 min in the chromosome of Escherichia coli: Psdh sdhCDAB-Psuc sucAB-sucCD. The gene encoding the lipoamide dehydrogenase component of both the 2-oxoglutarate and pyruvate dehydrogenase complexes (E3; IpdA) is the distal gene of another cluster containing two promoters located at 2 · 7 min: Ppdh pdhR-aceEF-PIpd IpdA. The responses of the suc and Ipd promoters to different environmental conditions and to regulator defects were investigated with appropriate IacZ fusions, in order to understand how expression of the sucAB genes is co-regulated with other genes in the sdhCDAB-sucABCD cluster and with IpdA expression. Expression from the suc promoter was repressed by IHF and partially activated by s38 but it was not regulated by ArcA, FNR, CRP, FruR or Fis, and not repressed by glucose or anaerobiosis, indicating that the well-established catabolite and anaerobic repression of ODH synthesis is imposed elsewhere. In contrast, the Ipd promoter was repressed by both glucose (via a CRP-independent mechanism) and anaerobiosis (mediated by ArcA), and activated by Fis, but it was not regulated by FNR, FruR, IHF or s38. These observations support the view that transcription of the sucABCD genes is primarily initiated and regulated at the upstream sdh promoter, and that the Ipd promoter is independently co-regulated with Psdh (primarily by ArcA-mediated repression) rather than with Psuc suc Direct evidence for co-transcription of the entire sdhCDAB-sucABCD region from Psdh was obtained by detecting a 10 kb transcript in rnc and rne mutants, but not in the parental strains. Three RNaseIII-specific processing sites, which contribute to the extreme instability of the readthrough transcript, were identified in the sdhCDAB-sucABCD intergenic region. Other sites of endonuclease processing were located by interpreting the patterns of transcript subfragments observed in Northern blotting.
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The gerC locus of Bacillus subtilis, required for menaquinone biosynthesis, is concerned only indirectly with spore germination
More LessThe gerC region of Bacillus subtilis comprises a tricistronic operon, encoding enzymes that catalyse the late stages of menaquinone biosynthesis. The gerC58 mutation is responsible for a severe growth defect; unsuppressed mutant cells grow as very short rods, which sometimes septate aberrantly. Cultures grow only to a low cell density, rapidly lose viability, and never sporulate. Unlinked suppressor mutations can restore near-normal growth. Several independent suppressed isolates were examined; all grew to normal cell length, but they showed, to varying extents, a residual defect in the placement of the cell division septum. The germination properties of the suppressed derivatives varied from normal to significantly slow in germination in all germinants; therefore, the combination of the gerC mutation and different suppressor alleles resulted in spores with very different germination properties. This suggests that any relationship between the gerC gene products and spore germination is indirect. The gerCC58 mutation maps in a gene encoding the catalytic subunit of the heptaprenyl-diphosphate synthase, which is responsible for formation of the isoprenoid side chain of menaquinone-7, and it is proposed that the gerCA, gerCB and gerCC genes be renamed hepA, menG and hepB, respectively.
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Glutamate and cyclic AMP regulate the expression of galactokinase in Mycobacterium smegmatis
More LessIt was found that Mycobacterium smegmatis is unable to utilize galactose as the sole carbon source because the sugar alone cannot induce galactokinase. However, galactokinase was induced by glutamate alone, and was further stimulated by galactose. Rifampicin completely inhibited the glutamate-mediated expression of galK in both the absence and presence of galactose. Extracellular cAMP stimulated the expression of the enzyme only in the presence of glutamate plus galactose. The galK gene from M. smegmatis, including its upstream promoter region, was cloned in a plasmid in Escherichia coli. The expression of kinase from these clones in E. coli was dependent on cAMP and its receptor protein (CRP). The expression of UDP-galactose 4-epimerase was constitutive. This and other evidence suggests that the galK gene is not linked to galT and galE in the mycobacterial genome. In a glutamate-independent galactose-utilizing mutant (gin-1 mutant) of M. smegmatis, galK was expressed in the absence of both galactose and glutamate, while in the presence of galactose this expression was increased twofold in the absence of glutamate and fourfold in its presence. Extracellularly added cAMP reduced the expression of the enzyme in the presence of galactose plus glutamate nearly to the basal level. It is proposed that in M. smegmatis the galK gene is expressed from two different promoters; the expression from one promoter is dependent on glutamate but not on galactose and cAMP, while that from the other requires all three components. The role of galactose is possibly to derepress the latter promoter.
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Cloning and heterologous expression of the gene for BLIP-II, a -lactamase-inhibitory protein from Streptomyces exfoliatus SMF19
More LessA -lactamase-inhibitory protein (BLIP-II) was purified from the culture filtrate of Streptomyces exfoliatus SMF19 and its N-terminal amino acid sequence was determined. A clone containing the gene encoding BLIP-II (bliB) was selected from a cosmid library by colony hybridization using an oligonucleotide probe based on the N-terminal amino acid sequence of BLIP-II. The bliB gene was isolated and sequenced. Analysis of the nucleotide sequence revealed that the gene consists of 1116 bp and encodes a mature protein of 332 amino acids preceded by a 40 amino acid signal sequence. bliB, expressed under the control of the T7 promoter in Escherichia coli, was accumulated in an inactive form in inclusion bodies, but -lactamase-inhibitory activity was recovered after refolding. In addition, bliB was heterologously expressed in Streptomyces lividans TK24 using the me/C1 promoter. The BLIP-II protein produced in recombinant strains of S. lividans was secreted into the culture supernatant in a biologically active form.
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Lysogenic bacteriophage M1 from Selenomonas ruminantium: isolation, characterization and DNA sequence analysis of the integration site
More LessBacteriophage M1 from the ruminal bacterium Selenomonas ruminantium strain ML12 comprises a 30 nm icosahedral capsid, a 25 nm tail and 48 kb of linear dsDNA with cohesive ends. A restriction map of the phage genome has been constructed. The presence of bacteriophage M1 in the rumen has been demonstrated by PCR amplification and Southern blot analysis of DNA from rumen bacterial samples obtained from ten different sheep. Lysogeny was demonstrated by hybridization of M1 DNA to host chromosomal DNA and by identification and cloning of a 2.3 kb region of the phage containing the predicted attP domain which promotes chromosomal integration. DNA sequencing of the attP region demonstrated two major ORFs surrounding the predicted attP site and structural analysis of this region revealed a motif comprising three different inverted repeats surrounding a 12 bp palindrome. Analysis of the translated amino acid sequence upstream of the attP site demonstrated the presence of conserved residues found within integrase proteins of several temperate phages of different bacterial species.
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Temporal transcription of the lactococcal temperate phage TP901-1 and DNA sequence of the early promoter region
More LessTranscriptional analysis by Northern blotting identified clusters of early, middle and late transcribed regions of the temperate lactococcal bacteriophage TP901-1 during one-step growth experiments. The latent period was found to be 65 min and the burst size 40.10. The eight early transcripts, all mapping in a 13 kb region adjacent to the attachment site of TP901-1, were present at maximal levels 10 min after infection. The four middle transcripts, observed at maximal levels 30 min after infection, are all located within a 2 kb region at the distal end of the early transcripts. The late class of transcripts were detected 40 min after infection and the amounts of these transcripts increased with time. The late transcripts were localized to the 13 kb region adjacent to the 2 kb middle transcribed region. The sequence of almost 4 kb of the early region was determined, allowing a detailed transcriptional map for the early region of which in total 6.4 kb was sequenced. Sequence analysis of the early region revealed two closely positioned but divergently orientated promoters, PL and PR, in accordance with the orientation of the ORFs and the transcriptional map. Nine ORFs were found, and similarities to a phage repressor, a single-stranded DNA-binding protein, a topoisomerase, a Cro-like protein and two other phage proteins of unknown function were detected. The gene arrangement in the early transcribed region of TP901-1 thus consists of two transcriptional units: one from PR containing four genes, of which at least two (the integrase gene and putative repressor) are needed for lysogeny, and the divergent and longer transcriptional unit from PL, presumably encoding functions required for the lytic life cycle. ORFs with homology to proteins involved in DNA replication were identified on the latter transcriptional unit.
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Dynamics of the pseudolysogenic response in slowly growing cells of Pseudomonas aeruginosa
More LessPseudolysogeny is an environmental condition in which the starved bacterial cell coexists in an unstable relationship with infecting viral genomes. As nutrients are supplied to the bacterium, the pseudolysogens resolve into either true lysogeny or active production of virions. The direct result of pseudolysogenic relationships is an extension of the effective phage half-lives in natural environments. In this paper a continuous culture model of interactions between bacterial host organisms and bacteriophages leading to pseudolysogeny is presented. The pseudolysogenic state was found to depend on the concentration of nutrients available to the host. As cells became more starved, the frequency of pseudolysogens increased. The dependence on overall nutrient concentration was more dramatic than the variation in the generation time (chemostat turnover time) of the host. Thus, it appears that pseudolysogeny is a legitimate strategy for environmental bacteriophages to adapt to survive periods of starvation of their host organisms. Consideration of pseudolysogeny as a survival strategy is important to the development of any comprehensive model of host-bacteriophage relationships in natural environments.
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Disruption studies of a Candida albicans gene, ELF1: a member of the ATP-binding cassette family
More LessA 3.6 kb gene (ELF1) with homology to the ATP-binding cassette (ABC) gene family has been isolated from genomic libraries of Candida albicans. Members of this gene family include both membrane transport proteins which confer a drug-resistance phenotype, and proteins whose functions are associated with protein translation. ELF1 (Elongation Like Factor) showed greatest homology with a Saccharomyces cerevisiae ORF (YPL226W), whose function is unknown, and lower homology with fungal elongation factor 3 (EF-3) genes. In comparison, homology with a gene conferring a drug-resistant phenotype (CDR1) was low. To understand the function of ELF1 in C. albicans, gene-knockout experiments were conducted using the hisG-URA3-hisG disruption cassette. Both single-copy (heterozygote) and double-disrupted strains in ELF1 were isolated. Phenotypically, the disrupted strains grew more slowly than wild-type and produced a mixture of large, irregular cells and apparently normal cells.
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Nitrate reduction and the isolation of Nit− mutants in Hansenula polymorpha
More LessHansenula polymorpha (syn. Pichia angusta) is able to grow on nitrate as sole nitrogen source. Nitrate reductase (NR) assays, optimized in crude extracts from nitrate-grown cells, revealed that NR preferentially used NADPH, but also used NADH, as electron donor and required FAD for maximum activity. NR activity was present in nitrate-grown and nitrite-grown cells, and was absent in cells grown in ammonium, glutamate and methylamine. Addition of reduced nitrogen compounds to nitrate-grown cells led to loss of NR activity, even if added with nitrate. Under nitrogen starvation, NR activity was not observed; however, following growth on nitrate, NR activity is maintained in the absence of nitrate. Increases but not decreases in NR activity were dependent on protein synthesis. Conditions for chlorate selection were optimized, and Nit− (nitrate−) mutants were isolated. Some of these mutants showed reduced or absent NR activity. Sixty-one NR− mutants revealed the monogenic recessive nature of their lesions and were grouped in 10 complementation classes. These mutants will be used in gene cloning experiments aimed at identifying structural and regulatory elements involved in the first step of nitrate reduction.
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The Rhizopus oryzae secreted aspartic proteinase gene family: an analysis of gene expression
More LessRhizopus oryzae was shown to possess a secreted aspartic proteinase gene family (sap) of at least four members (sap1-sap4). Within the family there was 77-87% identity at the nucleotide level and 76-92% identity at the amino acid level. Transcription of three members of this gene family (sap1-sap3) required an acidic medium (pH<4.5) and either nitrogen or sulphur derepression. Regulation was co-ordinate and hierarchical, with pH occupying the higher position in the hierarchy. Exogenous protein increased transcript levels, probably via the provision of metabolic intermediates rather than by direct induction of gene expression. sap4 was not expressed under these conditions. SAP1-SAP4 are predicted to have almost identical substrate-binding sites and therefore substrate specificity. It is proposed that sap1-sap3 exist to provide amplified expression of the secreted aspartic proteinase because protein, an important secondary nitrogen source for this fungus, requires extensive degradation to make its nitrogen available to the cell.
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Intron polymorphism in small subunit rDNA of Nectria galligena
More LessPCR amplification of the small subunit (SSU) rDNA gene of 40 isolates of Nectria galligena revealed four length polymorphisms. PCR-RFLP analysis of the SSU rDNA gene divided the isolates into four categories similar, but not identical, to categories identified by Southern-RFLP analysis. Nucleotide sequence analysis revealed that isolates in three of the four SSU rDNA (18S) categories possess an intron of 363 bp, 1185 bp or 1423 bp at the NS 7 priming site. Isolates in the fourth category do not possess an intron. The nucleotide sequences of these introns did not contain the core elements characteristic of typical group I introns, nor did they exhibit a group I intron secondary structure. Homology between the introns indicates a common lineage, all three possibly having come from a larger intron and having been formed by subsequent deletions. PCR primers upstream of the SSU rDNA intron region and from within the internal transcribed spacer 1 region amplify a product specific to N. galligena, which will confirm the identity of the pathogen and reveal its 18S category in a single reaction.
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- Genomics
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Physical map of the chromosome of Aeromonas salmonicida and genomic comparisons between Aeromonas strains
More LessI-Ceul and Pmel physical maps of the Aeromonas salmonicida A449 chromosome were constructed using PFGE. The circular chromosome of A. salmonicida A449 was estimated to be 4658.30 kb. The approximate location of several genes, including those encoding proteins implicated in virulence, were identified. The map showed that the known virulence-factor-encoding genes were not clustered. The I-Ceul genomic digestion fingerprints of several typical and atypical strains of A. salmonicida were compared. The results confirmed the homogeneity of typical strains, which provided further support for the clonality of the population structure of this group. Extensive diversity was observed in the I-Ceul digestion fingerprint of atypical strains, although a clonality was observed in the strains isolated from diseased goldfish. The results suggest that comparison of I-Ceul digestion fingerprints could be used as a powerful taxonomic tool to subdivide the atypical strains and also help clarify some of the current confusion associated with the taxonomy of the genus Aeromonas.
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Physical-genetic map of the erythromycin-producing organism Saccharopolyspora erythraea
More LessA physical map of the chromosome of the erythromycin-producing actinomycete Saccharopolyspora erythraea NRRL 2338 has been constructed using the restriction enzymes Asel and Dral. The map was constructed by a variety of methods including linking clone analysis, cross-hybridizations using labelled macrorestriction fragments, gene probing, two-dimensional PFGE and restriction enzyme site generation. Analysis of the individual macrorestriction patterns of the 17 Asel-, 6 Dral- and 22 Asel/Dral-digested fragments indicated a chromosome size of about 8 Mb. Linking clones for five contiguous Asel fragments were obtained, covering 32% of the chromosome. The linkage of an additional eight Asel fragments was aided by the finding that the rRNA operons of S. erythraea contain an Asel site within the 16S (rrs) gene. Generation of S. erythraea strains that contain additional Dral sites within selected Asel fragments, followed by PFGE analysis and Southern hybridization to determine specific linkages, facilitated the completion of the Asel map. The entire Dral map was constructed by gene probing and cross-hybridizations. PFGE analysis of agarose-embedded DNA prepared in either the presence or absence of proteinase K suggested that the S. erythraea NRRL 2338 chromosome is linear. A total of 15 genes or gene clusters were mapped to specific Asel and Dral fragments, including the erythromycin-biosynthetic gene cluster and the rRNA operons.
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- Pathogenicity And Medical Microbiology
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Site-directed mutagenesis of streptococcal plasmin receptor protein (Plr) identifies the C-terminal Lys334 as essential for plasmin binding, but mutation of the plr gene does not reduce plasmin binding to group A streptococci
More LessPlasmin(ogen) binding is a common property of many pathogenic bacteria including group A streptococci. Previous analysis of a putative plasmin receptor protein, Plr, from the group A streptococcal strain 64/14 revealed that it is a glyceraldehyde-3-phosphate dehydrogenase and that the plr gene is present on the chromosome as a single copy. This study continues the functional characterization of Plr as a plasmin receptor. Attempts at insertional inactivation of the plr gene suggested that this single-copy gene may be essential for cell viability. Therefore, an alternative strategy was applied to manipulate this gene in vivo. Site-directed mutagenesis of Plr revealed that a C-terminal lysyl residue is required for wild-type levels of plasmin binding. Mutated Plr proteins expressed in Escherichia coli demonstrated reduced plasmin-binding activity yet retained glyceraldehyde-3-phosphate dehydrogenase activity. A novel integration vector was constructed to precisely replace the wild-type copy of the plr gene with these mutations. Isogenic streptococcal strains expressing altered Plr bound equivalent amounts of plasmin as wild-type streptococci. These data suggest that Plr does not function as a unique plasmin receptor, and underscore the need to identify other plasmin-binding structures on group A streptococci and to assess the importance of the plasminogen system in pathogenesis by inactivation of plasminogen activators and the use of appropriate animal models.
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Rickettsia Rickettsii Infection of the Ea.Hy 926 Endothelial Cell Line: Morphological Response To Infection and Evidence for Oxidative Injury
More LessEA.hy 926 is a permanent human cell line that expresses highly differentiated functions characteristic of human vascular endothelium. Rickettsia rickettsii can efficiently infect and cause a cytopathic effect in EA.hy 926 cells. R. rickettsii produced visible lytic plaques in EA.hy 926 cells at 10 d postinfection (p.i.) following application of a secondary agarose overlay containing 2 μg emetine ml-1 and 40 μg NaF ml-1 on day 2. Rickettsial growth in EA.hy 926 cells had a similar profile to that occurring in human umbilical vein endothelial cells (HUVEC) and rickettsiae catalysed polymerization of actin tails. Intracellular multiplication of R. rickettsii resulted in significant changes in the internal morphology of EA.hy 926 cells, most notably extensive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope by 72 h p.i. These events correlated with significant alterations in the host-cell antioxidant system, including decreased levels of intracellular reduced glutathione and glutathione peroxidase activity and increased amounts of intracellular peroxide through to 96 h of infection. These findings are similar to the changes described previously for R. rickettsii-infected HUVEC and suggest that common mechanisms associated with rickettsia-induced oxidative injury occur in the two cell lines. EA.hy 926 cells were also used to investigate the influence of the antioxidant α-lipoic acid on rickettsial infection. Overnight pretreatment with 1-500 μM α-lipoic acid did not prevent cells from being destroyed following infection with rickettsiae. Supplementation of the culture medium with 1 and 10 μM α-lipoic acid 2 h after rickettsial inoculation also did not provide any protective effect. However, 100, 200 and 500 μM α-lipoic acid increased the viability of infected cells at 96 h to 45, 51 and 70%, respectively compared with 26% for untreated, infected samples. Thiol levels and glutathione peroxidase activity in treated, infected cells increased and peroxide content decreased proportionally to increasing α-lipoic acid concentrations. Furthermore, treatment with 500 μM α-lipoic acid for 72 h p.i. completely prevented ultrastructural changes in infected cells. In conclusion, the permanent endothelial cell line EA.hy 926 is susceptible to injury induced by R. rickettsii infection. Although the cellular changes resulting from infection are not identical in all aspects to that demonstrated previously in HUVEC, the increased reproducibility and convenience of EA.hy 926 cells make them suitable for biochemical and morphological studies.
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16S rRNA gene sequences of ‘Candidatus Campylobacter hominis’, a novel uncultivated species, are found in the gastrointestinal tract of healthy humans
More LessAlthough some Campylobacter species are agents of gastroenteritis and periodontal disease in humans, little is known of the variety of campylobacters in the gastrointestinal tract of healthy individuals. This paper provides evidence for the existence of a previously undescribed, uncultivated Campylobacter species that may be a commensal in the healthy human gut. Saliva and faeces from 20 healthy individuals were examined by PCR assays specific for nine species of campylobacter (C. sputorum, C. concisus, C. upsaliensis, C. helveticus, C. Iari, C. fetus, C. hyointestinalis, C. jejuni and C. coli) and for the genus as a whole. Genus-specific amplicons were produced from 19 of 20 saliva samples and from 18 of 20 faecal samples. C. concisuss species-specific amplicons were produced from 19 of 20 saliva samples and 3 of 20 faecal samples. The faecal samples were all PCR-negative for other Campylobacter species. Three unidentified 16S rRNA Campylobacter genus-specific amplicons of faecal origin were sequenced. Phylogenetic analysis showed that these sequences were 99% similar, and clustered within the genus as a novel group which was termed HS (HS = healthy subject). A PCR primer pair specific for the HS group was designed from the sequence data and used to reexamine the original samples. Although it was not possible to culture the organism from faeces, specific PCR assay detected it in 10 of the 20 faecal samples, but not in any corresponding saliva samples. The authors propose that the source of the amplicons is a previously undescribed and so far uncultivated species, which they term ‘Candidatus Campylobacter hominis’.
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Phototrophic oxidation of ferrous iron by a Rhodomicrobium vannielii strain
More LessOxidation of ferrous iron was studied with the anaerobic phototrophic bacterial strain BS-1. Based on morphology, substrate utilization patterns, arrangement of intracytoplasmic membranes and the in vivo absorption spectrum, this strain was assigned to the known species Rhodomicrobium vannielii. Also, the type strain of this species oxidized ferrous iron in the light. Phototrophic growth of strain BS-1 with ferrous iron as electron donor was stimulated by the presence of acetate or succinate as cosubstrates. The ferric iron hydroxides produced precipitated on the cell surfaces as solid crusts which impeded further iron oxidation after two to three generations. The complexing agent nitrilotriacetate stimulated iron oxidation but the yield of cell mass did not increase stoichiometrically under these conditions. Other complexing agents inhibited cell growth. Ferric iron was not reduced in the dark, and manganese salts were neither oxidized nor reduced. It is concluded that ferrous iron oxidation by strain BS-1 is only a side activity of this bacterium that cannot support growth exclusively with this electron source over prolonged periods of time.
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Evaluation of the intranasal challenge route in mice as a mucosal model for Candida albicans infection
More LessThe intranasal route was used to study Candida albicans infections in mice. Mice from two different inbred strains were challenged intranasally with C. albicans and the level of local and systemic colonization was monitored. DBA/2 mice were highly susceptible to challenge and viable C. albicans disseminated from the lungs to deeper tissues, including kidneys, liver and spleen within 48 h. In contrast, in BALB/c mice challenged in the same manner, C. albicans were retained within the lungs and cleared. Local and systemic anti-C. albicans immune responses were investigated. BALB/c mice exhibited higher titres of serum and mucosal anti-C. albicans IgA than DBA/2 mice. Splenocytes from BALB/c mice, but not from DBA/2 mice, produced detectable levels of interleukin-4 and -5 following stimulation with C. albicans antigens. Both DBA/2- and BALB/c-derived splenocytes produced interferon-γ and interleukin-10 in response to similar stimulation. In conclusion, the intranasal route provided a simple, non-invasive murine model for investigating C. albicans infection through mucosal surfaces.
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Overexpression of Candida albicans secretory aspartyl proteinase 2 and its expression in Saccharomyces cerevisiae do not augment virulence in mice
More LessTo elucidate the implications of secreted aspartyl proteinase (Sap)2p in the pathogenesis of Candida infections, the SAP2 gene was expressed in Saccharomyces cerevisiae and overexpressed in Candida albicans. The coding region of SAP2, including its signal sequence and propeptide, was amplified by PCR and cloned downstream of the S. cerevisiae or C. albicans ADH1 promoter. Plasmid expression of SAP2 in S. cerevisiae showed that the signal peptide was functional. Integrative transformation of S. cerevisiae and C. albicans was accomplished by homologous recombination within the URA3 locus for S. cerevisiae and the SAP2 locus for C. albicans. Negative control transformants carried plasmids either without the SAP2 insert or with mutated sap2. S. cerevisiae and C. albicans transformants showed similar growth rates to their parental strains or negative controls, when grown in medium containing amino acids. However, in medium with BSA as sole nitrogen source, constitutive expression of SAP2 enabled S. cerevisiae to grow and increased the growth rate of C. albicans. In both media, only S. cerevisiae transformants harbouring SAP2 secreted the enzyme, as confirmed by proteinase activity assays and immunoblotting. When C. albicans was grown in amino acids medium, the enzyme was detected exclusively in transformants constitutively expressing SAP2. However, in BSA medium these strains secreted enzyme earlier and secreted higher amounts of enzyme and total proteinase activity. In pathogenicity studies in intact mice, expression of Sap2p as a sole putative virulence factor did not cause S. cerevisiae to become virulent and constitutive overexpression of SAP2 did not augment virulence of C. albicans in experimental oral or systemic infection.
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- Physiology And Growth
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Green fluorescent protein as a novel species-specific marker in enteric dual-species biofilms
More LessGreen fluorescent protein (GFP) was used as a tool to examine the interactions between pairs of bacterial species and their effects on subsequent biofilm development over 24 h. A plasmid encoding GFP from Aequorea victoria was transformed into strains of Enterobacter agglomerans and Escherichia coli ATCC 11229. The development of dual-species biofilms, containing one fluorescent and one non-fluorescent partner, was examined using viable counts. UV illumination of plates enabled both species to be identified in a mixture. The spatial distribution of each species was examined by UV microscopy, simultaneously staining the non-fluorescent strain with propidium iodide. GFP fluorescence was measured to quantify the adhesion of the strains to other cells or cell constituents or the invasion into pre-existing biofilms. Cooperation between Ent. agglomerans/GFP and Klebsiella pneumoniae G1 resulted in a 54 and a 23% increase in biofilm formation, respectively, compared with single-species biofilms. E. coli/GFP and Serratia marcescens 87b stably co-existed in biofilms but did not affect the growth of each other. The other bacterial partnerships examined were competitive, with the end result that one species dominated the biofilm. The methods described provide a convenient technique for the examination of mixed-species biofilm communities where the unique interactions between species determine the true properties of the resultant biofilms.
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Membrane-bound and extracellular β-lactamase production with developmental regulation in Streptomyces griseus NRRL B-2682
More LessA new type ofβ-lactamase has been isolated and characterized in Streptomyces griseus NRRL B-2682. The enzyme has membrane-bound and extracellular forms. Biochemical characterization of some of the properties of the enzyme showed that it belongs to the class A group of penicillinases. Comparison of the membrane-bound and extracellular forms of theβ-lactamases suggests that they seem to be differently processed forms of the same enzyme. The N-terminal amino acid sequence of the extracellular form of the β-lactamase showed a high degree of similarity to a D-aminopeptidase of another Streptomyces griseus strain. Secretion of the β-lactamase was affected by the differentiation state of the strain since in spontaneous non-sporulating mutants only the membrane-bound form was present. In accordance with this when sporulation of the wild-type strain was inhibited it failed to secrete extracellular β-lactamase. Addition of globomycin to the non-sporulating cells liberated the enzyme from the membrane, indicating that the protein is processed normally by signal peptidase II and a glyceride-thioether group, together with a fatty acid amide-linkage, is responsible for the attachment of the enzyme to the cellular membrane. Under sporulation-repressed conditions addition of peptidoglycan fragments and analogues or inhibition of cell wall biosynthesis by penicillin-G induced β-lactamase secretion and also restored sporulation both in solid and submerged cultures. These results confirm that β-lactamase secretion is tightly coupled to the sporulation process in S. griseus.
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Attacin - an insect immune protein - binds LPS and triggers the specific inhibition of bacterial outer-membrane protein synthesis
More LessAttacin is a 20 kDa antibacterial protein, originally isolated from the immune haemolymph of Hyalophora cecropia. It has been demonstrated previously that attacin causes increased permeability of the outer membrane of Escherichia coli and inhibition of outer-membrane protein synthesis at the transcriptional level. This is accompanied by inhibition of growth. Here, LPS is shown to serve as the receptor for attacin and evidence is presented that attacin does not need to enter the cell to exert its activity. The increase in outer-membrane permeability precedes any increase in inner-membrane permeability by at least one generation time (∼ 45 min), and the inhibiting effect of attacin on synthesis of outer-membrane proteins is detectable after only 10 min. It is also shown that attacin causes induction of several stress proteins and increased synthesis of LPS within, respectively, 25 and 60 min of treatment. Based on the results presented, it is proposed that attacin has the unique ability to specifically interfere with synthesis of outer-membrane proteins without entering the inner membrane or cytoplasm.
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Excess production of phage λ delayed early proteins under conditions supporting high Escherichia coli growth rates
Bacteriophage λ is unable to lysogenize Escherichia coli hosts harbouring the rpoA341 mutation due to a drastic reduction in transcription from CII-activated lysogenic promoters (p E, p 1 and p aQ). In addition, the level of early transcripts involved in the lytic pathway of λ development is also decreased in this genetic background due to impaired N-dependent antitermination. Here, it is demonstrated that despite the reduced level of early lytic p L- and p R-derived transcripts, lytic growth of bacteriophage λ is not affected in rich media. The level of the late lytic, p R-derived transcripts also remains unaffected by the rpoA341 mutation under these conditions. However, it was found that whilst there is no significant difference in the phage burst size in rpoA + and rpoA341 hosts growing in rich media, phage λ is not able to produce progeny in the rpoA341 mutant growing in minimal medium, in contrast to otherwise isogenic rpoA + bacteria. Provision of an excess of the phage replication proteins O and P in trans or overproduction of the antitermination protein N restore the ability of phage λ to produce progeny in the rpoA341 mutant under the latter conditions. These results suggest that in rich media phage λ produces some early proteins in excess of that needed for its effective propagation and indicate that replication proteins may be limiting factors for phage lytic growth in poor media.
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- Systematics And Evolution
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Identification and sequencing of the groE operon and flanking genes of Lawsonia intracellularis: use in phylogeny
Proliferative enteropathy (PE) is a complex of diseases of commercial importance to the pig industry. The obligate intracellular bacterium Lawsonia intracellularis is consistently associated with PE and pure cultures of this bacterium have been used to reproduce PE in pigs. In this study L. intracellularis bacteria were purified directly from PE-affected tissue. DNA extracted from purified bacteria was used to construct a partial genomic library which was screened using sera from L. intracellularis-immunized rabbits. Two seroreactive recombinant clones were identified, one of which expressed proteins of 10 and 60 kDa. The sequence of the insert from this clone, plSI-2, revealed ORFs with sequence similarity to the groES/EL operon of Escherichia coli, the 50S ribosomal proteins L21 and L27 of E. coli, a GTP-binding protein of Bacillus subtilis and a possible protoporphyrinogen oxidase, HemK, of E. coli. Primers designed from unique sequences from the plSI-2 insert amplified DNA from infected, but not non-infected, porcine ilea; the amplicon sequence obtained from tissue-cultured L. intracellularis was identical to the corresponding sequence in plSI-2, confirming the origin of the clone. The sequence of L. intracellularis GroEL and other GroEL sequences in the databases were used to construct a partial phylogenetic tree. Analysis of the GroEL sequence relationship suggested that L. intracellularis is not significantly related to other organisms whose GroEL sequences are held in the databases and supports previous data from 16S sequence analyses suggesting that L. intracellularis is a member of a novel group of enteric pathogens.
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