- Volume 141, Issue 10, 1995
Volume 141, Issue 10, 1995
- Review Article
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- Microbiology Comment
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- Environmental Microbiology
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The use of two-dimensional gradient plates to investigate the range of conditions under which conjugal plasmid transfer occurs
More LessSummary: Gel-stabilized two-dimensional gradient plates were used to study the effects of pH, salt concentration and temperature on the conjugal transfer of plasmid RP4 between strains of Escherichia coli and Pseudomonas putida. The combinations of pH and salt concentration that permitted conjugation were mapped as a two-dimensional growth area occupied by transconjugants following conjugation. This conjugation domain was less extensive than the areas that supported growth of the parental strains, and showed evidence for the interactive effects of pH and salt concentration in determination of conditions that permitted conjugation. The size and shape of the conjugation domain was influenced by time, temperature, the identities of the donor and recipient bacteria, and the combination of donor and recipient bacteria.
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- Genetics And Molecular Biology
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Pilus biogenesis gene, pilC, of Neisseria gonorrhoeae: pilC1 and pilC2 are each part of a larger duplication of the gonococcal genome and share upstream and downstream homologous sequences with opa and pil loci
More LessSummary: Pili of Neisseria gonorrhoeae mediate attachment of the bacteria to target cells and undergo both phase and antigenic variation. PilC is a 110 kDa minor pilus-associated protein involved in pilus biogenesis and attachment. The expression of PilC is turned on and off at high frequency and is controlled by frameshift mutations in a run of G residues positioned in the region encoding the signal peptide. Most strains of N. gonorrhoeae carry two copies of pilC. The DNA sequence of pilC1 of strain MS11 is presented and compared to the sequence of the 3′ end of pilC2. These two genes are highly homologous, but not identical. The putative transcriptional terminator of pilC1 contains a pair of inverted uptake sequences for gonococcal DNA (5′-GCCGTCTGAA-3′). An 88 bp sequence located upstream of the pilC1 gene has also been reported to precede several opa genes of N. gonorrhoeae. Shorter regions positioned both downstream and upstream of pilC1 can also be found in silent pil loci as well as close to opa genes. The pilC genes are part of a duplication of a larger DNA region extending more than 2 kb downstream of the coding region.
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In Bacillus subtilis 168, teichoic acid of the cross-wall may be different from that of the cylinder: a hypothesis based on transcription analysis of tag genes
More LessSummary: Five of the genes known to encode the enzymes for the synthesis of poly(glycerol phosphate), the major teichoic acid of Bacillus subtilis 168, are organized in two divergently transcribed operons, tagAB and tagDEF. lacZ and gus transcriptional fusions to the first genes of these operons revealed that: (i) in media of different richness, higher growth rates were paralleled by lower transcription levels; (ii) upon transition to stationary phase, the transcription per unit mass of both operons increased abruptly by a factor of about two; and (iii) a rise in temperature was accompanied by decreased transcription of tagA and increased transcription of tagD. Mapping of transcription start points revealed two divergent aA-controlled promoters. Although tagD and the neighbouring downstream gene tagE are transcribed from the same promoter, the latter was expressed at a much lower level than the former. Moreover, expression of tagE, and of the translationally coupled tagF, did not increase at the onset of the stationary phase, indicating that additional regulatory signals may act in the intergenic tagD-tagE region. Optimal transcription of these operons appears to require the entire regulatory region, suggesting that tag gene expression may, among other factors, be regulated by the three-dimensional configuration of this segment. The biological implications of these results are discussed.
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Glucosaminidase of Bacillus subtilis: cloning, regulation, primary structure and biochemical characterization
More LessSummary: The 90 kDa glucosaminidase protein was purified to apparent homogeneity from vegetative cells of Bacillus subtilis AC327, and then the corresponding gene was cloned into Escherichia coli in two inactive forms by standard procedures. Nucleotide sequencing of the glucosaminidase region revealed a monocistronic operon, (designated lytD = cwlG) encoding a 95.6 kDa protein, comprising 880 amino acid residues, which has a typical signal peptide. Moreover, another monocistronic operon (designated pmi = orfX), encoding a 35.4 kDa protein, was found upstream of the glucosaminidase gene. Expression of a lytD-lacZ fusion gene, driven by lytD regulatory sequences, was observed during the exponential growth phase. The introduction of a sigD null mutation greatly reduced (by about 95%) the expression of the fusion. Amino acid sequence analysis of the glucosaminidase showed two types of direct repeats, each type being present twice, in the N-terminal-to-central region of the glucosaminidase: these repeats probably represent the cell-wall-binding domain. Zymographic analysis revealed that the 90 kDa glucosaminidase is partly processed to several smaller proteins (35-39 kDa), retaining lytic activity. Processing of these proteins occurred between the N-terminal cell-wall-binding and C-terminal catalytic domains of the glucosaminidase, the site being located between the 569th and 606th codons of the glucosaminidase. Serial deletions from the N-terminus of the glucosaminidase revealed that the loss of more than one repeating unit drastically reduces its lytic activity toward cell walls. The lytD gene product, in either an intact or a truncated form, was found to be lethal for E. coli, and the N-terminally truncated glucosaminidase proteins, produced in E. coli, were very unstable. The partially purified glucosaminidase from B. subtilis was found to be very unstable at low ionic strength at 37 °C, but this instability was overcome by the addition of either SDS-purified cell wall or protease inhibitor (PMSF) to the enzyme or after purification of the glucosaminidase to apparent homogeneity.
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Sequence, genetic analysis, and expression of Actinobacillus pleuropneumoniae transferrin receptor genes
More LessSummary: The tbpA and tbpB genes encoding the transferrin receptor proteins Tbp 1 and Tbp2 from a serotype 7 strain of Actinobacillus pleuropneumoniae were cloned, sequenced, and expressed in Escherichia coli. The tbpB gene was preceded by putative promoter and regulatory sequences and was separated from the downstream tbpA gene by a 13 bp intercistronic sequence suggesting that the two genes may be coordinately transcribed. Determination of the nucleotide sequence of this region facilitated PCR amplification of the tbp region from a serotype 1 strain for comparative purposes. The deduced amino acid sequences of the Tbp1 proteins had regions of homology with Neisseria Lbp and Tbp1s and with TonB-dependent outer membrane (OM) receptors of E. coli. The deduced amino acid sequences of the Tbp2 proteins were nearly identical to those presented in previous studies. Upon high-level expression of the tbpA gene, a large proportion of the recombinant Tbp1 was found in inclusion bodies and could not be affinity-isolated with immobilized porcine transferrin. Most of the remaining expressed Tbp1 was present in the OM fraction, was expressed at the surface of E. coli cells, and retained binding activity that was specific for the C-lobe of porcine transferrin. Although recombinant Tbp2 was found in inclusion bodies during high-level expression, a significant proportion was associated with a novel OM fraction that appeared in sucrose density gradients which was distinct from the OM fraction containing recombinant Tbp1. The recombinant Tbp2 was accessible at the surface yet was unable to bind porcine transferrin. In contrast to previous observations, the binding by recombinant Tbp2 was specific for the C-lobe of porcine transferrin. These results indicate that the A. pleuropneumoniae transferrin receptor proteins have similar properties to the receptor proteins in Neisseria spp. and Haemophilus influenzae, and that functional studies performed with recombinant receptor proteins need to consider differences in processing and export of these proteins when expressed in heterologous hosts.
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Two heterocyst-specific DNA rearrangements of nif operons in Anabaena cylindrica and Nostoc sp. strain Mac
More LessSummary: Two site-specific DNA rearrangements occur during heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120: the deletion of an 11 kb element from within the nifD gene and the deletion of a 55 kb element from within the fdxN gene. Three Nostoc and six Anabaena strains were screened for the presence of the nifD and fdxN elements by Southern hybridization with Anabaena PCC 7120 DNA probes. Eight of the nine strains contained DNA sequences that were similar to the nifD element. Three strains, Nostoc sp. strain Mac, Anabaena cylindrica and Anabaena sp. strain M131, also showed significant similarity to portions of the 55 kb fdxN element. Anabaena sp. strain CA lacked both the nifD and fdxN elements. Southern analysis of vegetative cell and heterocyst DNA from A. cylindrica and a Fox+ revertant of Nostoc Mac (isolate R2) showed rearrangement of the nifD and fdxN elements in heterocysts. We found no RFLPs between Anabaena M131 and Anabaena PCC 7120 suggesting that strain M131 is a Het- derivative of strain PCC 7120.
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The CrP operon of Chlamydia psittaci and Chlamydia pneumoniae
More LessSummary: One of the critical developmental events during the unique intracellular life cycle of Chlamydiae is their differentiation from a metabolically active, replicative form or reticulate body (RB) to an infectious extracellular form of the organism (elementary body or EB). This process is characterized by the expression of two extraordinarily cysteine-rich envelope proteins of molecular masses 9 kDa and 60 kDa. We describe the molecular cloning and sequence determination of the 9 kDa cysteine-rich proteins (CrPs) of C. pneumoniae and C. psittaci. Comparison of these 9 kDa CrP amino acid sequences with those of C. trachomatis showed regions of structural variation and conservation. Transcription of the 9 kDa CrP genes occurred as both a monocistronic message and as a bicistronic message which included the 60 kDa CrP gene. Transcription of the 9 kDa and 60 kDa CrP genes was tightly linked to the chlamydial growth cycle with synthesis of their mRNAs and consequent translation of the 60 kDa CrPs occurring as RBs differentiated to form EBs. The maximal rate of transcription occurred late in the growth cycle from a single but highly conserved promoter which had close similarity with the Escherichia coli consensus promoter sequences. A stem and loop structure which could be involved in regulating translation of mRNA occurred in all three species between the transcriptional start point and the ribosome binding site. Although transcription is initiated from a single promoter in all three chlamydial species, transcriptional termination points for the monocistronic and bicistronic mRNAs differ in both number and position.
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Sequence and functional analysis of the Streptomyces phaeochromogenes plasmid pJV1 reveals a modular organization of Streptomyces plasmids that replicate by rolling circle
Summary: pJV1 is an 11 kb, high-copy-number conjugative Streptomyces phaeochromogenes plasmid that replicates by the rolling circle mechanism (RCR). Sequencing combined with functional analysis of deletion, insertion and frameshift mutations was used to characterize the genes involved in plasmid transfer and chromosome mobilization (Cma), the single-strand origin for RCR and an associated strong incompatibility (Sti) determinant. pJV1 contains two essential transfer genes whose expression is regulated by an adjacent repressor gene with similarity to the GntR family of regulators. A consensus sequence specific for the helix-turn-helix motifs of repressor proteins of Streptomyces plasmids is proposed. Unregulated expression of the transfer genes by inactivation of the repressor is lethal. Three additional genes increase intramycelial plasmid spread resulting in pock formation but, unlike the essential transfer genes, are not required for Cma. The pJV1 transfer genes and their regulatory region, but not the minimal replication region encoding the double-strand replication origin and replication protein, are similar in their sequence and arrangement to those of the Streptomyces nigrifaciens plasmid pSN22, revealing a modular organization of Streptomyces RCR plasmids.
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Depression of streptomycin production by Streptomyces griseus at elevated growth temperature: studies using gene fusions
More LessSummary: Streptomyces griseus ATCC 12475 fails to produce streptomycin when grown at 34 °C or above, although growth is appreciable up to at least 37 °C. This depression of streptomycin production at elevated growth temperature is manifest equally in liquid and on solid, and with complex and minimal, media. We report studies with gene fusions of the reporter genes aph or xylE to restriction fragments containing the streptomycin biosynthesis promoter PstrB1. aph constructs were in high, and xylE constructs in low, copy number vectors. Two strB1 promoter fragments were used, one requiring activation by the pathway-specific activator StrR of S. griseus, the other reportedly activator independent. PstrB1 expression in the aph constructs in S. griseus and in S. lividans was significantly reduced at 37 °C compared to 30 °C. Some of this reduction could be explained by lower plasmid copy number at the higher temperature, but strR-dependent expression was clearly temperature controlled. Using the xylE reporter system, the temperature dependence of PstrB1 expression was confirmed but, surprisingly, the strR dependence of the two promoter fragments differed from that observed in the multicopy aph constructs. These data identify a temperature-dependent promoter which may contribute to the depressive effect of elevated growth temperature on streptomycin production.
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The tuf3 gene of Streptomyces coelicolor A3(2) encodes an inessential elongation factor Tu that is apparently subject to positive stringent control
More LessSummary: In Streptomyces coelicolor A3(2), two genes, tuf1 and tuf3, encode the apparent polypeptide chain elongation factors EF-Tu1 and EF-Tu3, respectively. While tuf1 appears to code for the major EF-Tu, the function of tuf3 is unknown. To assess the role of EF-Tu3, tuf3 was subjected to mutational and transcriptional analyses. Replacement of the 5′-half of tuf3 by an antibiotic resistance cassette had no detectable effect on phenotype, indicating that tuf3 is not essential for growth or differentiation. The transcription start site of tuf3 was located approximately 195 nt upstream of the translation start site. The sequence of the tuf3 promoter (Ptuf3) resembles the consensus for the major class of eubacterial promoters, and Ptuf3 was recognized preferentially by an RNA polymerase fraction enriched in αrdB, the principal . factor of S. coelicolor. Nuclease S1 mapping failed to reveal tuf3 transcripts during growth of S. coelicolor in liquid culture, consistent with the apparent absence of EF-Tu3 in total protein extracts of the same strain. However, tuf3 transcription was observed in cultures of S. coelicolor M145 shortly after nutritional shiftdown (which resulted in the disappearance of tuf1 transcripts) and after addition of serine hydroxamate, both of which induce the stringent response. Transcription of tuf3 was also observed in transition-phase and stationary-phase cultures of S. coelicolor J1681, a strain deleted for bldA (which specifies a tRNALeu for the rare leucine codon UUA). In all of these examples, transcription of tuf3 followed the production of ppGpp, consistent with the hypothesis that tuf3 is subject to positive stringent control.
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Response of the bvg regulon of Bordetella pertussis to different temperatures and short-term temperature shifts
More LessSummary: Bordetella pertussis produces a number of virulence factors whose expression is coordinately regulated by the bvgAS locus. Transcription of virulence genes is repressed by environmental factors such as low temperature (25°C) and chemical stimuli. Temperature shift of bacterial cultures from 25°C to 37°C activates two classes of bvg-regulated virulence genes: the early genes, which are activated within 10 min, and late genes, which require 2-4 h for activation. During the interval between the activation of the early and late genes, the intracellular concentration of BvgA increases 50-fold. It has been proposed that this increased concentration may be required for the activation of the late genes. Here we have analysed the response of the bvg locus to intermediate temperatures and to repeated temperature shifts. Temperature shifts of B. pertussis cultures from 22°C to 28°C, 32°C or 35°C resulted in the synthesis of low, intermediate, and high amounts of BvgA. This implied that the intracellular concentration of BvgA is temperature-dependent. We have also observed that the amount of virulence factors produced correlates with the BvgA concentration. When bacteria grown at 37°C were shifted to 22 °C, transcription from the adenylate cyclase toxin haemolysis promoter (PAC) was repressed after 30 min, while transcription from the bvg (P1) and filamentous haemagglutinin (PFHA) promoters was repressed after 2 h. During this time, the amount of BvgA did not decrease. A subsequent temperature shift from 22°C to 37°C induced transcription from the P, and PFHA promoters after 10 min and transcription from the PAC promoter after 20 min. This result shows that in the presence of a high concentration of BvgA, the time lag between temperature shift and late promoter transcription is reduced from 2-4 h to 20 min. The above data support the proposal that the concentration of BvgA plays a role in activating expression of the late genes.
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A 17 kDa outer-membrane protein (Omp4) from Serratia marcescens confers partial resistance to bacteriocin 28b when expressed in Escherichia coli
More LessSummary: A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli and clones were screened for a bacteriocin 28b insensitive phenotype. One clone was found that showed partial resistance to bacteriocin 28b. By using Tn5tac1 insertions it was shown that this phenotype was due to the expression in E. coli of an outer-membrane protein of 17 kDa (Omp4). The DNA region defined by insertion mutagenesis was sequenced and found to contain an ORF of 515 bp. The deduced amino acid sequence has 172 residues with a theoretical molecular mass of 18.4 kDa. The protein contains an N-terminal signal sequence of 24 amino acid residues and, when compared to other enterobacterial outer-membrane proteins, most closely resembles a family of small outer-membrane proteins of Enterobacteriaceae whose known functions appear to be related with virulence. Immunoblotting experiments showed that Omp4 is present in 15 biotypes of S. marcescens. The bacteriocin 28b resistance phenotype conferred on E. coli by Omp4 appears to be pleiotropic since overexpression of the Omp4-encoding gene leads to a decrease in the amount of OmpA, OmpF and/or OmpC; OmpA and OmpF are the receptors for bacteriocin 28b in E. coli.
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Nucleotide sequence of the mxcQ and mxcE genes, required for methanol dehydrogenase synthesis in Methylobacterium organophilum XX: a two-component regulatory system
More LessSummary: Nucleotide sequence analysis of the mxcQ and mxcE loci, required for the synthesis of methanol dehydrogenase in Methylobacterium organophilum XX, has revealed two open reading frames that show significant similarity to sequences of prokaryotic two-component systems, especially MxaY and MxaX proteins of another methylotrophic bacterium, Paracoccus denitrificans. Cell-free extracts and DNA-column-fractionated proteins from wild-type M. organophilum XX cells grown on methanol or succinate contained protein(s) that were able to bind specifically to the upstream region of methanol dehydrogenase large subunit gene (mxaF). In contrast, cell-free extracts from mxcQ and mxcE mutant strains of M. organophilum XX had zero or reduced binding activity towards the promoter fragments of the mxaF gene. This is consistent with the involvement of the mxcQ and mxcE genes in transcriptional regulation of methanol dehydrogenase synthesis. Analyses of sequential deletions of the mxaF upstream region have defined the functional boundary of the promoter/operator region of this gene and identified one nucleotide segment as essential to the activation of mxaF.
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Poly--hydroxybutyrate (PHB) biosynthetic genes in Rhizobium meliloti 41
More LessSummary: Genes encoding -ketothiolase (phaA), acetoacetyl-CoA reductase (phaB) and PHB-synthase (phaC) from R. meliloti 41, together with a fourth gene, referred to as ORF1, presumed to be involved in PHB biosynthesis, have been cloned and sequenced. phaA, phaB and ORF1 were identified by heterologous hybridization on a cosmid library, while phaC was isolated by cloning the transposon-tagged fragment from a R. meliloti PHB- Tn5 mutant. phaA and phaB were functionally expressed in Escherichia coli while phaC was able to complement a PHB- strain of R. meliloti 41. The three genes were sufficient to direct the production of polyhydroxyalkanoate in E. coli. The homology of ORF1 with an ORF located near the PHB genes in two phototrophic bacteria suggests its involvement in PHB synthesis.
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An isoleucyl-tRNA synthetase gene from Campylobacter jejuni
More LessSummary: A complete isoleucyl-tRNA synthetase gene (ileS) of Campylobacter jejuni was isolated from a C. jejuni TGH9011 genomic DNA library constructed in pBluescript. The complete coding sequence, flanking regions and transcription start point were determined. The deduced isoleucyl-tRNA synthetase (lleRS) had 917 amino acids with a molecular mass of 105399 Da, which was consistent with the observed size of 105 kDa in Escherichia coli maxicells. The ileS gene was mapped onto the physical map of the C. jejuni genome. Alignment of the C. jejuni lleRS sequence with six other bacterial lleRS sequences and two lower eukaryotic lleRS sequences identified seven conserved motifs, including the two signature sequences, HIGH and KMSKS, of class I aminoacyl-tRNA synthetases.
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Molecular analysis of an extracellular protease gene from Vibrio parahaemolyticus
More LessSummary: The structural gene prtVp encoding the extracellular protease of Vibrio parahaemolyticus strain 93 was cloned in Escherichia coli and sequenced. The cloned DNA fragment contained a 1761 bp ORF encoding a 587 amino acid protein. The deduced polypeptide is composed of a 25 amino acid signal peptide and a 562 amino acid extracellular polypeptide with a calculated molecular mass of 63 156 Da. Protease analysis using a gelatin-containing SDS-polyacrylamide gel detected the presence of a 62 kDa protease that was present in the culture supernatant fractions of the wild-type V. parahaemolyticus strain and of E. coli bearing a pUC119 recombinant with the prtVp DNA insert. The protease activity was inhibited by zinc- and metal-specific inhibitors such as EDTA and 1,10-phenanthroline, which suggested that it is a metalloprotease. The deduced amino acid sequence of PrtVp has 32% identity with that of the collagenase of Vibrio alginolyticus, but has no identity with those of the bacterial proteases. A conserved zinc-binding domain was also found in PrtVp from homology comparison with other metalloproteases. This PrtVp can cause weak haemolysis on blood agar.
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Charaterization of cryptic prophages (monocins) in Listeria and sequence analysis of a holin/endolysin gene
More LessSummary: Monocins in Listeria were induced by UV-irradiation of liquid cultures, and defective phage particles were purified from the lysates. Electron microscopy showed flexible, non-contractile bacteriophage-tail-like particles, consisting of specific proteins of molecular mass 20-45 kDa and pl 4.6-6.7. These particles were able to lyse listerial cells. DNA sequence homologies between chromosomal DNA of monocin-producing strains and labelled Listeria phage DNAs were inferred from DNA/DNA hybridizations, suggesting that most of the prophage DNA is still present in the listerial chromosome. An endolysin gene cpl2438 was cloned from listerial chromosomal DNA and was identified by its expression of lytic activity against Listeria cells in a bioassay. The gene consists of 864 nt encoding a protein of 287 aa with a calculated molecular mass of 32975 Da (CPL2438). This is in good agreement with the size of a protein observed in SDS-PAGE after overexpression of the lytic protein in Escherichia coli. The nucleotide sequence of a putative holin gene (hol2438, 291 nt) upstream of cpl2438 was determined after PCR-amplification of listerial DNA and it shows typical features common to the holin gene family. Expression of the encoded protein (HOL2438, 95 aa, 10.1 kDa) in E. coli was found to be lethal for the host cells. The results underline the close relationship between monocins and intact Listeria bacteriophages, indicating that monocins are incompletely assembled phage particles derived from cryptic prophages of Listeria, probably including the phage lysin.
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A plasmid encoding enzymes for nylon oligomer degradation: nucleotide sequence and analysis of pOAD2
More LessSummary: The entire nucleotide sequence of nylon oligomer degradative plasmid pOAD2 from Flavobacterium sp. KI723T1 was determined. pOAD2 comprises 45519 bp, with a 66.6 mol% G + C content. The precise loci of the four nylon oligomer degradation genes, namely nylA (6-aminohexanoate-cyclic-dimer hydrolase gene), nylB) (6-aminohexanoate-dimer hydrolase), nylB' (a gene having 88% homology to nylB) and nylC (endo-type 6-aminohexanoate oligomer hydrolase), and five IS6100 elements were identified on this plasmid. Comparison of the sequence of pOAD2 with those in the GenBank and EMBL databases revealed that the deduced amino acid sequences from eight regions of pOAD2 had significant similarity with the sequences of gene products such as oppA-F (oligopeptide permeases), ftsX (filamentation temperature sensitive), penDE (isopenicillin N-acyltransferase) and rep (plasmid incompatibility). A functional map of pOAD2 is presented.
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The DNA and RNA polymerase genes of yeast plasmid pGKL2 are essential loci for plasmid integrity and maintenance
More LessSummary: Novel recombinant plasmids derived from the Kluyveromyces lactis killer plasmid k2 have been constructed to study plasmid biology and gene function. In vivo recombination between native resident k2 and suitable disruption vectors, employing the KlTRP1 gene fused to a plasmid promoter as selection marker, yielded ORF2 and ORF6 deletion plasmids at high frequencies. As judged from Southern hybridization and plasmid restriction mapping analyses, these novel hybrids, termed rk2/2 and rk2/6, respectively, carry deletions in their putative DNA (ORF2) and RNA (ORF6) polymerase structural genes with central regions replaced by the input marker DNA. Long-term selection for TRP1 over 350 generations of growth did not favour maintenance of hybrids over wild-type k2. Thus, neither rk2/2 nor rk2/6 was fully functional and able to displace parental k2, indicating that both target genes are essential for plasmid integrity or maintenance. Recombinant plasmids were reduced in copy number relative to k2 with rk2/2 more drastically affected than rk2/6 implying a direct involvement of the ORF2 product in plasmid replication and an indirect maintenance function for the ORF6 gene product.
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Isolation of canCHS1A, a variant gene of Candida albicans chitin synthase
Summary: A canCHS1A gene encoding the chitin synthase of Candida albicans was cloned. DNA sequencing and comparison with another canCHS1 gene described elsewhere indicated that the canCHS1A gene encoded a polypeptide with 775 amino acid residues, a protein with one less amino acid than that encoded by the canCHS1 gene. A six-base alteration was observed between the two genes, suggesting that the canCHS1A gene is a variant gene of the canCHS1. The pH profile/activity relationship of canChs1A in permeabilized cells was identical to that of canChs1. The canChs1A enzyme was competitively inhibited by polyoxin D (5.2 μM) and nikkomycin Z (12 μM). When the cloned gene was expressed in a Saccharomyces cerevisiae chs2 mutant that exhibited aberrant morphology, the normal structure was restored. We conclude that the function of the canCHS1A gene is similar to that of sacCHS2 in S. cerevisiae.
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- Pathogenicity And Medical Microbiology
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Mapping of β-1,2-linked oligomannosidic epitopes among glycoconjugates of Candida species
More LessSummary: The distribution of β-1,2-linked oligomannosides among glycoconjugates of various Candida species was investigated by Western blotting, using monoclonal and polyclonal antibodies which react with these epitopes. Expression of β-1,2-linked oligomannosidic epitopes on a 14-18 kDa polydisperse antigen nonreactive with concanavalin A (ConA), previously identified as a C. albicans serotype A phospholipomannan (PLM), appeared to be restricted to C. albicans serotypes A and B (including var. C. stellatoidea types I and II) and C. tropicalis. In C. albicans, β-1,2-linked oligomannosidic epitopes also appeared to be slightly associated with high molecular mass (> 100 kDa) polydisperse ConA-reactive mannoproteins. For all the other Candida strains investigated, belonging to the species C. parapsilosis, C. krusei, C. glabrata and C. robusta (S. cerevisiae), β-1,2-linked oligomannosidic epitopes were found to be present in association with medium molecular mass (18-100 kDa) and high molecular mass ConA-reactive mannoproteins, giving reproducible labelling profiles that varied between species.
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Aspergillus fumigatus antigens
More LessSummary: Cytosolic fractions of mycelial extracts from Aspergillus nidulans, A. flavus, and three different isolates of A. fumigatus, grown to stationary phase in Czapek-Dox-AOAC medium, were tested by immunoblotting for the presence of antigens reactive to 80 serum samples from aspergilloma patients. Fifty control serum samples were used to determine the specificity of the reactions. In the A. fumigatus cytosolic fraction a group of four main antigenic bands (p90, p60, p40 and p37) was consistently recognized (in total or partial form) by 90% of the serum samples from the aspergilloma patients. This group of antigens was designated as the ‘cytosolic fraction complex’ (CFC). As confirmed by two-dimensional electrophoresis followed by immunoblotting with aspergilloma serum samples, each of the four antigenic bands is formed of several isoforms of acidic glycopeptides with slightly different pls. All the isoforms are at least N-glycosylated, as demonstrated by endoglycosidase H removal of a considerable amount of sugar residues. The relationship of these antigens with certain other A. fumigatus antigens previously reported in the literature, and their potential use in the immunodiagnosis of aspergilloma, are discussed.
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A highly immunogenic putative Mycobacterium kansasii lipoprotein
More LessSummary: The resurgence of tuberculosis, the emergence of multiple drug resistant tuberculosis, and the increasing prevalence of mycobacterial disease in AIDS patients have increased the importance of defining new mycobacterial antigens that can be utilized in the development of improved diagnostic reagents and more effective vaccines. In this report, a highly immunogenic Mycobacterium kansasii protein (MK35) and the gene encoding this antigen were characterized. MK35 gene probes reacted with genomic DNA from M. avium, M. bovis BCG, M. intracellulare and M. tuberculosis but not with DNA isolated from nine other mycobacterial species. Nucleotide sequence analysis showed that the MK35 gene encodes a 26 kDa protein which contains a consensus bacterial lipoprotein processing sequence. In addition, detergent-phase separation studies strongly suggested that MK35 is a lipoprotein. Skin test assays demonstrated that MK35 elicited a strong response in guinea pigs sensitized with M. kansasii but did not react in M. tuberculosis-sensitized guinea pigs. These results further suggest that mycobacterial lipoproteins are immunogenic antigens that should be considered in the development of new mycobacterial vaccines and diagnostic reagents.
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Polypeptides associated with tufts of cell-surface fibrils in an oral Streptococcus
More LessSummary: Cells of the oral bacterium Streptococcus oralis CN3410 produce lateral tufts of cell-surface fibrils of two lengths. Treatment of cells with trypsin resulted in loss of the tufts and release of longer fibrils intact. SDS-PAGE analysis of trypsin extracts containing fibrils revealed two groups of high molecular mass polypeptides which were denoted group A (molecular mass 227-246 kDa) and group B (molecular mass 175-208 kDa). Antibodies were raised to these two groups of trypsin-extracted polypeptides (TEPs) and to purified fibrils, and the reactivities of the three different antisera were found to be similar both on nitrocellulose blots of cell-surface polypeptides and in ELISA with whole cells. Similar patterns of TEPs were obtained from cells of a spontaneously derived mutant strain, KP34V, which lacked the short fibril components of tufts. Cells of strain KP34V had similar cell-surface hydrophobicity to strain CN3410 cells, and adhered to the same extent to parotid salivary pellicle or human buccal epithelial cells (BECs) as the wild-type cells. Trypsin treatment of strain CN3410 cells abolished their surface hydrophobicity and ability to adhere to BECs, but did not affect streptococcal cell binding to experimental salivary pellicle. Antibodies to TEPs or fibrils had no effect on cell adhesion to BECs or salivary pellicle. The results imply that the short fibril components of tufts are not involved in the cell adhesion properties tested. It is suggested that the TEPs are components of long fibrils, but they are not determinants of streptococcal cell adhesion to pellicle or to epithelial cells.
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Ribosomal internal transcribed spacer sequences are identical among Mycobacterium avium-intracellulare complex isolates from AIDS patients, but vary among isolates from elderly pulmonary disease patients
More LessSummary: Sequencing 280 bp of the internal transcribed spacer (ITS) between the 16S and 23S rRNA genes in a collection of 46 clinical isolates of the Mycobacterium avium-intracellulare complex (MAI complex) identified nine different sequences, grouping these isolates in nine ‘ITS sequevars’. This analysis extends the subdivision within the MAI complex to 18 ITS sequevars and also improves discrimination from other mycobacterial species. Evaluation of the sequevar grouping among different clinical sources revealed strong association of the M. avium sequevar Mav-B with AIDS and with lymphadenitis in children (18 out of 20 and 3 out of 3 respectively). Isolates from elderly patients with pulmonary disease and not suspected of being HIV infected belonged predominantly to M. intracellulare ITS sequevars and sequevars not assigned to either M. avium or M. intracellulare. On the other hand, animal isolates were of both the Mav-A and Mav-B sequevars. We conclude that ITS sequevar typing is an accurate way of identifying distinct MAI complex strains. The observed differences between clinical sources suggest that ITS sequevars reflect possibly important, biologically and clinically relevant polymorphisms between MAI complex organisms.
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Role of LPS length in clearance rate of bacteria from the bloodstream in mice
Summary: Strains of Pseudomonas aeruginosa isolated from patients with cystic fibrosis (CF) never spread systemically. This may be due to serum sensitivity since these strains are very sensitive to complement-mediated bactericidal activity. A serum-resistant mutant, P. aeruginosa TUM3 HSR, was obtained from serum-sensitive strain TUM3 from a CF patient in order to clarify the mechanism of failure of systemic spread. LPS profiles on silver-stained gels and immunological analysis revealed that a long O-polysaccharide side chain was overproduced on the LPS molecules of TUM3 HSR as compared with the LPS of TUM3. The clearance rate from the bloodstream in mice was compared in the two strains. The number of TUM3 bacteria in 1 ml of blood, 10 min after injection into the tail vein, significantly decreased from 1.7 × 108 to 3.7 × 105 c.f.u. ml-1. In contrast, TUM3 HSR was not eliminated during the same period (decrease from 1.9 × 108 to 3.4 × 107c.f.u. ml-1). Interestingly, these isogenic strains were not killed by 40% murine serum, probably reflecting immaturity of the complement-mediated killing system in mice. These results pointed to a correlation between LPS structure and blood clearance rate in mice. This was confirmed by examining blood clearance kinetics using the smooth-LPS strain Salmonella typhimurium LT2 and LPS-deficient mutants derived from it. S. typhimurium LT2 resisted blood clearance while the LPS-deficient mutants were cleared rapidly. None of the S. typhimurium strains were killed by murine serum. The number of P. aeruginosa TUM3 and S. typhimurium LPS-deficient mutants trapped in the liver following injection into the peripheral circulation was greater than that of their counterparts. These results indicate that the long O-polysaccharide side chain of LPS may play a crucial role in evading phagocytosis by the reticuloendothelial system (RES), and therefore, may control the establishment of systemic infection by Gram-negative bacteria. The interaction between complement C3 on bacteria and C3 receptors on macrophages may also be involved in the trapping mechanism by the RES. It was expected that the level of C3 bound on the cell surface would be higher in P. aeruginosa TUM3 or the LPS-deficient mutants derived from S. typhimurium LT2. However, our flow-cytometric results demonstrated that the level of C3 was almost identical in isogenic strains.
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- Physiology And Growth
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Phosphatase production and activity in Citrobacter freundii and a naturally occurring, heavy-metal-accumulating Citrobacter sp.
More LessSummary: The ability of a naturally occurring Citrobacter sp. to accumulate cadmium has been attributed to cellular precipitation of CdHPO4, utilizing HPO2- 4 liberated via the activity of an overproduced, Cd-resistant acid-type phosphatase. Phosphatase production and heavy metal accumulation by batch cultures of this strain (N14) and a phosphatase-deficient mutant were compared with two reference strains of Citrobacter freundii. Only strain N14 expressed a high level of acid phosphatase and accumulated lanthanum and uranyl ion enzymically. Acid phosphatase is regulated via carbon-starvation; although the C. freundii strains overexpressed phosphatase activity in carbon-limiting continuous culture, this was approximately 20-fold less than the activity of strain N14 grown similarly. Citrobacter strain N14 was originally isolated from a metal-contaminated soil environment; phosphatase overproduction and metal accumulation were postulated as a detoxification mechanism. However, application of Cd-stress, and enrichment for Cd-resistant C. freundii (‘training’), reduced the phosphatase activity of this organism by about 50% as compared to Cd-unstressed cultures. The acid phosphatase of C. freundii and Citrobacter N14 had a similar pattern of resistance to some diagnostic reagents. The enzyme of the latter is similar to the PhoN acid phosphatase of Salmonella typhimurium described by other workers; the results are discussed with respect to the known phosphatases of the enterobacteria.
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A NADP-glutamate dehydrogenase mutant of the petit-negative yeast Kluyveromyces lactis uses the glutamine synthetase-glutamate synthase pathway for glutamate biosynthesis
Summary: The activities of the enzymes involved in ammonium assimilation and glutamate biosynthesis were determined in wild-type and NADP-glutamate dehydrogenase (GDH) null mutant strains of Kluyveromyces lactis. The specific NADP-GDH activity from K. lactis was fivefold lower than that found in Saccharomyces cerevisiae. The glutamine synthetase (GS) and glutamate synthase (GOGAT) activities were similar to those reported in S. cerevisiae. The NADP-GDH null mutant was obtained by transforming the uraA strain MD2/1 with a linearized integrative yeast vector harbouring a 390 bp fragment of the NADP-GDH structural gene. This mutant grew as well as the parent strain on ammonium, but showed GS and GOGAT activities higher that those found in the wild-type strain, implying that the GS-GOGAT pathway could play a leading role in glutamate biosynthesis in K. lactis. Southern blotting analysis of K. lactis chromosomes separated by contour-clamped homogeneous electric field electrophoresis, indicated that the NADP-GDH structural gene is localized on chromosome VI.
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Induction and repression of a-amylase production in batch and continuous cultures of Aspergillus oryzae
More LessSummary: The intra- and extracellular concentrations of a-amylase in Aspergillus oryzae have been measured during batch culture of a wild-type strain and two recombinant strains. The mean intracellular level for the two recombinant strains was about four to five times the level of the wild-type strain. The recombinant strains also had a higher a-amylase productivity, whereas the residence time of the intracellular a-amylase pool was approximately the same for the three strains. At high glucose concentrations there was a low constitutive synthesis of a-amylase, whereas at low glucose concentrations derepression resulted in an increased production rate. Shifts from a glucose- to a maltose-limited chemostat showed that maltose induces both the production and secretion of a-amylase. Finally, from immunoblots, both a glycosylated and an unglycosylated a-amylase have been detected.
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The role of alanine in the acute response of Giardia intestinalis to hypo-osmotic shock
More LessSummary: The effect of medium hypo-osmolality on cell volume and intracellular amino acid composition was studied in the protozoan parasite Giardia intestinalis. When G. intestinalis was exposed to hypotonic medium, it initially swelled by 40% of its original volume and then decreased its volume, thus demonstrating a regulatory volume decrease process (RVD). These processes were accompanied by a rapid release of intracellular neutral amino acids, especially alanine, but not by amino acids with net charges such as glutamate and ornithine. The net alanine efflux was Na+ and Cl- independent, and sensitive to medium osmolality. Alanine efflux was sigmoidal with respect to medium osmolality, with an approximately linear relationship over a range of 250 mOsm kg-1. Alanine efflux was also sensitive to temperature, and an Arrhenius plot gave a Q 10 of 3.6 and an activation energy of 25 kcal mol-1 (105 kJ mol-1), suggesting that a carrier-type transport protein, or uniport, was involved in the net alanine efflux under hypotonic conditions. This volume-activated (VA) alanine efflux was not inhibited by ionophores or chloride channel blockers. Of the potential inhibitors tested, only p-hydroxymercuribenzoate inhibited net alanine efflux. This thiol reagent also inhibited giardial RVD, suggesting that alanine efflux plays a significant role in this process. The VA (alanine) uniport was able to transport 2-aminoisobutyric acid (AIB), a structural analogue of alanine which is frequently used for the characterization of eukaryotic alanine transport, but which is not transported by Giardia under isotonic conditions. On the basis of AIB uptake under hypotonic conditions and lack of transactivation of AIB efflux from AIB-loaded cells by external 10 mM alanine or glycine under isotonic conditions, it is evident that the VA (alanine) uniport is different from the previously reported (alanine) antiport.
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Analysis of lysine-dependent yeast sporulation: a decrease in cyclic AMP is not required for initiation of meiosis and sporulation in Saccharomyces cerevisiae
More LessSummary: Cells of the yeast Saccharomyces cerevisiae sporulated in nutrient-rich medium containing l-lysine. Sporulation was specific to the presence of L-lysine and was initiated when the cellular content of this basic amino acid reached approximately 0·2-0·5 mmol (g cells)−1, at early stationary phase. The formation of asci was most efficient at pH 7·0 and 50-100 mM L-lysine; in these optimum conditions, the sporulation frequency reached about 60% after 5 d incubation. The L-lysine-dependent sporulation system in nutrient-rich conditions was distinct from the currently used potassium-acetate-dependent system in nutrient-deficient conditions. Analysis of the L-lysine-dependent system indicated that, prior to entrance into meiosis and/or sporulation processes, the yeast cells change in shape, their pool sizes for L-cysteine and glutathione alter, and they synthesize a protein with a molecular mass of 15 kDa. A low level of cAMP was not required for the entrance into meiosis and/or sporulation.
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Nitrogenase quantity varies diurnally in a subset of cells within colonies of the non-heterocystous cyanobacteria Trichodesmium spp.
More LessSummary: Trichodesmium is a marine filamentous cyanobacterium with the exceptional ability to fix atmospheric nitrogen during the day without differentiating the specialized oxygen-protective cells known as heterocysts. The localization of the Fe-protein of nitrogenase (dinitrogenase reductase) was examined in cross-sectioned colonies of three species: Trichodesmium thiebautii, T. tenue and T. erythraeum, using immunogold/transmission electron microscopy and immunofluorescence/light microscopy. The enzyme was confined to a limited subset of cells (on average 14%) in the sections, randomly distributed within the colonies in all three species. The frequency of nitrogenase-containing cells varied on a diurnal basis, being highest and comparatively constant during the day, the period of active nitrogen fixation. The percentage of nitrogenase-labelled cells decreased in the evening and approached zero just before dawn. After sunrise a period of rapid synthesis of nitrogenase followed. Although the frequency of nitrogenase-containing cells was constant during the day, the relative concentration of nitrogenase within the labelled cells clearly increased until noon and then successively decreased in the afternoon/night. The data imply that in three species of the non-heterocystous cyanobacterium Trichodesmium there are a limited number of cells, or possibly whole trichomes (filaments), which are specialized for nitrogen fixation. Most likely the same set of cells is active throughout the day, with the variation in activity being due to a variation in the quantity of nitrogenase within each cell. Comparisons between nitrogenase-containing Trichodesmium cells and heterocysts are discussed.
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Agrobacterium radiobacter and related organisms take up fructose via a binding-protein-dependent active-transport system
More LessSummary: Washed cells of Agrobacterium radiobacter prepared from a fructose-limited continuous culture (D 0.045 h-1) transported D-[U-14C]fructose in a linear manner for up to 4 min at a rate several-fold higher than the rate of fructose utilization by the growing culture. D-[U-14C]Fructose transport exhibited a high affinity for fructose (K T < 1 μM) and was inhibited to varying extents by osmotic shock, by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and by unlabelled sugars (D-fructose/D-mannose > D-ribose > D-sorbose > D-glucose/D-galactose/D-xylose; no inhibition by D-arabinose). Prolonged growth of A. radiobacter in fructose-limited continuous culture led to the selection of a novel strain (AR100) which overproduced a fructose-binding protein (FBP) and showed an increased rate of fructose transport. FBP was purified from osmotic-shock fluid using anion-exchange fast protein liquid chromatography (FPLC). The monomeric protein (Mr 34200 by SDS-PAGE and 37700 by gel-filtration FPLC) bound D-[U-14C]-fructose stoichiometrically (1.17 nmol nmol FBP-1) and with high affinity (KD 0.49 μM) as shown by equilibrium dialysis. Binding of D-[U-14C]fructose by FBP was variably inhibited by unlabelled sugars (D-fructose/D-mannose > D-ribose > D-sorbose; no inhibition by D-glucose, D-galactose or D-arabinose). The N-terminal amino acid sequence of FBP (ADTSVCLI-) was similar to that of several sugar-binding proteins from other species of bacteria. Fructose transport and FBP were variably induced in batch cultures of A. radiobacter by growth on different carbon sources (D-fructose > D-ribose/D-mannose > D-glucose; no induction by succinate). An immunologically similar protein to FBP was produced by Agrobacterium tumefaciens and various species of Rhizobium following growth on fructose. It is concluded that fructose is transported into A. radiobacter and related organisms via a periplasmic fructose/mannose-binding-protein-dependent active-transport system, in contrast to the phosphotransferase system used by many other species of bacteria.
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Long-chain haloalkanes are incorporated into fatty acids by Rhodococcus rhodochrous NCIMB 13064
More LessSummary: The fatty acid composition of the cellular lipids of Rhodococcus rhodochrous NCIMB 13064 grown on various long-chain haloalkanes has been investigated and the influence of halogen substituents, carbon chain length and the position of halogen substitution in the growth substrate explored. Of the total fatty acids present in cells grown on 1-chloro-, 1-bromo- and 1-iodohexadecane, 75, 90 and 81%, respectively, were substituted in the w position by the corresponding halogen but only 1% of the fatty acids present after growth on 1-fluorotetradecane were fluorinated in this position. The extent of the halofatty acid incorporation with different halogen substituents in the growth substrate appears to reflect the degree to which oxygenase attack is restricted to the non-halogenated end of the haloalkane. Studies of the fatty acid composition of cells after growth on a series of 1-chloroalkanes containing an even number of carbon atoms between C10 and C18 indicated chlorofatty acid incorporation from C12 to C18 substrates at levels ranging from 21% with C12 to 75% with C16. The chlorofatty acids formed by initial oxidation of the chloroalkane were chain-lengthened or chain-shortened by from two to eight carbon atoms, with accompanying desaturation in some instances. Substantial quantities of a methyl-branched C19:0 chlorofatty acid were also present with several chloroalkane substrates. When the fatty acid composition of cells after growth on 1-bromoalkanes containing an odd number of carbon atoms between C11 and C17 was examined, the incorporation of bromofatty acids was observed with C13, C15 and C17 substrates; a maximum of 76% was recorded for the C15 bromoalkane. As with even chain-length chloroalkanes, both chain-lengthening and -shortening occurred predominantly via two-carbon units so that most bromoacids present possessed an odd number of carbon atoms. When 1-bromododecane or 2-bromododecane were substrates, overall incorporations of bromofatty acids into the lipid fraction were very similar, demonstrating that the position of halogen substitution in the haloalkane was not critical in determining the extent of incorporation of the haloacids into cellular lipids. The results of the study indicate a mechanism by which degradation products of chlorinated paraffins could enter the biological food chain.
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Acetyl-CoA-dependent pyruvate carboxylase from the photosynthetic bacterium Rhodobacter capsulatus: rapid and efficient purification using dye-ligand affinity chromatography
More LessSummary: Pyruvate carboxylase (PC) was purified to homogeneity from an overexpressing strain of the purple photosynthetic bacterium Rhodobacter capsulatus using a rapid dye-ligand affinity chromatography procedure, in which dye-bound enzyme was specifically eluted with a low concentration of acetyl-CoA, an allosteric activator of the enzyme. The enzyme purified by this method was obtained in 75% yield with a specific activity of 40 U (mg protein)-1. In contrast, affinity chromatography on a monomeric avidin column, commonly used in the purification of biotin-containing carboxylases, resulted in a yield of < 40%, with a specific activity of 10 U (mg protein)-1 The enzyme purified by the dye-linked procedure had a subunit molecular mass of 140000 Da and was absolutely dependent on acetyl-CoA for activity. Acetyl-CoA was also effective in protecting the enzyme from thermal denaturation. The enzyme was inhibited by 2-oxoglutarate and, to a lesser extent, L-aspartate, with sigmoidal kinetics with respect to acetyl-CoA concentration. The amino acid composition, pH optimum and kinetic constants for pyruvate, ATP and bicarbonate were determined. An N-terminal sequence of 26 residues was obtained, which was homologous to the N-terminal regions of several eukaryotic PCs, propionyl-CoA carboxylases and acetyl-CoA carboxylase.
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A new chitosanase gene from a Nocardioides sp. is a third member of glycosyl hydrolase family 46
More LessSummary: Strain N106, a newly isolated soil actinomycete classified in the genus Nocardioides on the basis of its chemotaxonomy, produced an extracellular chitosanase and was highly active in chitosan degradation. A gene library of Nocardioides sp. N106 was constructed in the shuttle vector pFD666 and recombinant plasmids carrying the chitosanase gene (csnN106) were identified using the 5′-terminal portion of the chitosanase gene from Streptomyces sp. N174 as a hybridization probe. One plasmid, pCSN106-2, was used to transform Streptomyces lividans TK24. The chitosanase produced by S. lividans(pCSN106-2) is a protein of 29.5 kDa, with a pl 8.1, and hydrolyses chitosan by an endo-mechanism giving a mixture of dimers and trimers as end-products. N-terminal sequencing revealed that the mature chitosanase is a mixture of two enzyme forms differing by one N-terminal amino acid. The csnN106 gene is 79.5% homologous to the csn gene from Streptomyces sp. N174. At the amino acid level, both chitosanases are homologous at 74.4% and hydrophobic cluster analysis revealed a strict conservation of structural features. This chitosanase is the third known member of family 46 of glycosyl hydrolases.
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Uptake of amino acids by the parasitic, flagellated protist Trichomonas vaginalis
More LessSummary: HPLC techniques have been applied to study amino acid uptake and release by Trichomonas vaginalis under a variety of conditions. Studies on the growth of T. vaginalis in complex media and the survival of the parasite in simple media, with and without amino acids and/or maltose, have shown that the growth or survival of T. vaginalis is better in the presence of maltose than when it is absent, and that greater amounts of amino acids are consumed by T. vaginalis in the absence of maltose. The results are consistent with several amino acids, notably arginine, threonine, leucine and methionine, being used by T. vaginalis as energy substrates. T. vaginalis released alanine and glycine into the culture media, the excretion being greater in the presence of maltose. These studies have provided new data on the uptake and release of amino acids by T. vaginalis and pave the way for detailed analysis of key enzymes and the regulation of the pathways involved.
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A gene (sleC) encoding a spore-cortex-lytic enzyme from Clostridium perfringens S40 spores; cloning, sequence analysis and molecular characterization
More LessSummary: Antiserum was raised against a 31 kDa spore-cortex-lytic enzyme, which is released during germination of Clostridium perfringens S40 spores. Western blotting of dormant spore and vegetative cell fractions separated by SDS-PAGE indicated that the 31 kDa enzyme is spore-specific and that the enzyme in the dormant spore exists as a 36 kDa protein which has no cortex-lytic activity. A gene encoding the 31 kDa enzyme, sleC, was cloned into Escherichia coli using a synthetic oligonucleotide as a hybridization probe and the nucleotide sequence of the entire gene was determined. The N-terminal amino acid sequence of the 36 kDa protein was found in this reading frame, confirming that the 36 kDa protein is a pro-form of the 31 kDa enzyme. The deduced amino acid sequence indicated that the 31 kDa enzyme is produced as a precursor, comprising three portions; an N-terminal prepro-sequence (114 amino acid residues), a pro-sequence (35 amino acid residues) and a mature enzyme (289 amino acid residues). It is suggested that the 36 kDa pro-enzyme is non-covalently attached to the exterior of the cortex layer, and that the pro-form is processed to release the active enzyme during germination.
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Glycosylation of the flagellin of the polar flagellum of Azospirillum brasilense, a Gram-negative nitrogen-fixing bacterium
More LessSummary: The glycosylation of the flagellin of the polar flagellum of Azospirillum brasilense Sp7 is demonstrated in several ways: (1) by a decrease in apparent M r after chemical deglycosylation; (2) by sugar staining after SDS-PAGE; (3) by use of a sugar-specific monoclonal antibody in immunogold labelling coupled with transmission electron microscopy. In addition, two mutant flagellins are described; they differ in glycan composition when compared with the wild-type. The glycosylation of a bacterial flagellin is unusual and has to be taken into account in the proposed model for the assembly of the flagellar filament.
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The isolation of isoagathenediol: a new tricyclic diterpene from the lipids of Rhodospirillum rubrum
More LessSummary: A tricyclic diterpene, isoagathenediol, has been isolated from the lipid fraction of the photosynthetic non-sulphur bacterium Rhodospirillum rubrum. The compound was present in trace amounts and its structure was determined by NMR spectroscopy and mass spectrometry. Isoagathenediol is a new natural product and may function as a membrane fluidity modulator. Five other bacterial species have also been screened for the presence of this compound, but the diterpene was detected only in extracts derived from R. rubrum. As tricyclic diterpene fossils are readily detected in oil and shale samples, the isolation of isoagathenediol has indicated the biological origin of these fossils.
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Glucose-induced, cyclic-AMP-independent signalling pathway for activation of neutral trehalase in the fission yeast Schizosaccharomyces pombe
More LessSummary: The addition of glucose to derepressed cells of Schizosaccharomyces pombe provokes a cAMP signal and activation of the cytoplasmic neutral trehalase. This transduction pathway does not require functional RAS protein since RAS1-disrupted cells exhibited a glucose response similar to that shown by control cells. Treatment of activated trehalase by alkaline phosphatase resulted in enzyme deactivation suggesting that trehalase may be modulated in vivo by reversible phosphorylation through cAMP-dependent protein kinase (PKA1). However, the addition of glucose to derepressed growing cells of Schiz. pombe lacking the catalytic subunit of protein kinase A (?pka1::URA4+ strains) induced stimulation of trehalase as well as phosphorylation of the enzyme protein. This glucose-induced response was absent in PKA1-deficient cells from resting cultures. Addition of exogenous cAMP activated trehalase in normal growing cells but failed to produce any effect on trehalase in PKA1-disrupted growing cells. These results confirm the occurrence of a PKA1-dependent pathway for trehalase activation and imply the existence of another glucose-induced phosphorylation pathway capable of activating trehalase during growth by a distinct, cAMP-independent protein kinase. At least one of the upstream components playing a role in the transduction of this alternative signal is either absent or inactive in cells from stationary phase and sporulated cultures. Cells harbouring the disrupted PKA1 gene responded also to a heat-shock signal by increasing trehalase activity, thus revealing that this enzyme may be a target common to various signalling pathways in the fission yeast.
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Gelatin fragments block adherence of Candida albicans to extracellular matrix proteins
More LessSummary: The adherence of Candida albicans to extracellular matrix proteins may be a critical step in the pathogenesis of candidiasis. Yeast cell adherence to type I and IV collagen, fibronectin and laminin was blocked by peptide fragments from denatured type I collagen (gelatin). Gelatin fragments were obtained by digestion of the reduced protein with trypsin or CNBr. The fragments did not have antifungal properties, presumably inhibiting adherence by blocking receptors (adhesins) on the surface of the fungus. A 10-mer (GQRGVVGLPG) fashioned from the a-1 chain of type I collagen reduced adherence by 68%. However, a gelatin peptide possessing 47 amino acids reduced fungal adherence to type I collagen by 100%. Peptides derived from the biocompatible protein gelatin, therefore, may have a potential role in reducing the adherence of the fungus to host proteins.
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Oestrogen-binding protein in Candida albicans: antibody development and cellular localization by electron immunocytochemistry
More LessSummary: Candida albicans, the most common fungal pathogen of humans, possesses an oestrogen (estrogen)-binding protein (EBP) that binds oestrogens with high affinity and specificity. The gene that encodes the EBP (CaEBP1) has been cloned and sequenced and shown to be structurally related to the old yellow enzyme from Saccharomyces cerevisiae. Here, we report the purification and the subcellular localization of the EBP from C. albicans. Using ion-exchange chromatography and an oestradiol affinity column, the EBP was purified from a strain of C. albicans (strain 422) which was selected because it constitutively expressed elevated levels of the binding protein. The purified protein displayed a subunit molecular mass of approximately 46 kDa when examined by denaturing gel electrophoresis, which is consistent with the size estimated from the sequence of the cloned CaEBP1 gene. An immunoaffinity column, prepared using a polyclonal antisera generated against EBP, depleted the oestrogen-binding activity from C. albicans cell extracts. Western blot analysis showed that the antisera specifically recognized the EBP from C. albicans. The antibodies also recognized the protein when the cloned CaEBP1 gene was expressed in S. cerevisiae and did not cross react with S. cerevisiae proteins. Using electron microscopy and antigen detection by immunogold staining, the EBP appeared to be primarily associated with vacuoles. However, when overexpressed in S. cerevisiae, the EBP was found diffusely throughout the cell. In conclusion, the EBP has been purified from C. albicans and antibodies generated against the protein were used to demonstrate that EBP is found associated with vacuoles in C. albicans.
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Unsaturated fatty acids are the active molecules of a glucan-synthase-inhibitory fraction isolated from entomophthoralean protoplasts
More LessSummary: A few entomophthoralean species are able to multiply in a protoplast form. The polysaccharide synthases which synthesize the cell wall are inactivated in this form. An inhibitor of one of the key enzymes of wall synthesis, glucan synthase, was isolated from entomophthoralean protoplasts, using silica column chromatography and HPLC. Thin-layer and gas chromatography revealed free fatty acids in the inhibitory fractions. These fatty acids, including long-chain unsaturated fatty acids, were shown to be responsible for the inhibition of glucan synthase. The fatty acids were generated during incubation of a protoplast homogenate for 36 h at 37 °C and were shown to be non-competitive and non-specific inhibitors of glucan synthase.
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- Plant-Microbe Interactions
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High frequency of conjugation versus plasmid segregation of RP1 in epiphytic Pseudomonas syringae populations
More LessSummary: The maintenance and transfer of the broad host-range plasmid RP1 in epiphytically growing populations of Pseudomonas syringae was monitored in the phyllosphere of bush bean (Phaseolus vulgaris). When foliage was inoculated with plasmid-containing bacteria, the plasmid was lost from the majority of the cells within 2 d but was stably maintained in 0.8% of the population. A high frequency of conjugation between added donors and recipients was observed under high humidity conditions. In 1 d, the number of transconjugants rose to 10-1 of the donors and the proportional level of transconjugants continued to increase until 3 d after inoculation. Under these conditions the proportion of plasmid-containing bacteria stabilized at about 0.8% of the total population. The conjugation rate appeared to be in equilibrium with plasmid loss and the slower growth of the plasmid-carrying cells. A factor that influenced the high conjugation frequency observed was the available nutrients provided by the leaf and also, to a lesser extent, the leaf surface itself. Transfer of the plasmid from added donors to indigenous bacteria was also studied, using a donor-specific bacteriophage for counterselection of the donor. Transfer was observed to 10 different species of Gram-negative epiphytically growing bacteria. The bean leaf surface appears to function as a hotspot at least for intraspecific transfer of plasmids in high humidity. The frequency of transfer was higher than in soil or in rhizosphere habitats. This is likely to be the result of an environment that is nutritionally rich in combination with a limited colonizable surface area which permits close contact between the bacterial cells.
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- Genome Analysis
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Physical and genetic map of the genome of Campylobacter upsaliensis
More LessSummary: A physical map of Campylobacter upsaliensis ATCC 43954 was constructed from DNA fragments generated by SalI (5′ G/TCGAC), NarI (5′ GG/CGCC) and BssHII (5′ G/CGCGC) restriction digests separated using pulsed-field gel electrophoresis. The size of the C. upsaliensis genome was approximately 2000 kb, providing evidence of the largest Campylobacter genome sized to date. Twenty-one fragments created from these restriction digests were assembled into a physical map using a combination of complementary methods including cross-Southern hybridization, hybridization fingerprint analysis and hybridization with homologous and heterologous (from Campylobacter jejuni) gene probes. The position of ten genetic loci, including that of the iron-uptake regulatory (fur) gene, were localized to the physical map. A genomic library of C. upsaliensis ATCC 43954 was constructed in lambda Gem-11 vector. Fifty thousand recombinants with an average size of 16 kb represent a library about 200 times the size of the genome. Using C. jejuni DNA probes, clones representing C. upsaliensis flaA, fur and ftsZ genes were isolated and localized to the physical map.
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Study of the organization of the genomes of Escherichia coli, Brucella melitensis and Agrobacterium tumefaciens by insertion of a unique restriction site
Summary: Tn5Map, a Tn5 derivative containing the 18 bp I-Scel site, was delivered from a RP4-mobilizable, RK6-derived suicide vector to Escherichia coli HB101, Brucella melitensis and Agrobacterium tumefaciens C58, which all lack natural I-Scel sites in their genomes. Digestion of the DNA from Tn5Map-containing strains and analysis by pulsed-field gel electrophoresis (PFGE) revealed that these derivatives contained a single transposon insertion. These digests also gave direct and independent proof for the single circular chromosome of E. coli, and for the presence of two circular chromosomes in B. melitensis and of a circular and a linear chromosome in A. tumefaciens C58 (which also contains two large circular plasmids). This rapid and versatile technique is potentially applicable to the study of the genomic organization in all Gram-negative bacteria which support Tn5 transposition. Moreover, linearization of circular replicons could be the first step for a rapid method of physical mapping.
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Volumes and issues
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Volume 86 (1975)
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Volume 85 (1974)
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Volume 84 (1974)
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Volume 83 (1974)
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Volume 82 (1974)
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Volume 81 (1974)
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Volume 80 (1974)
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Volume 79 (1973)
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Volume 78 (1973)
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Volume 77 (1973)
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Volume 76 (1973)
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Volume 75 (1973)
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Volume 74 (1973)
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Volume 73 (1972)
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Volume 72 (1972)
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Volume 71 (1972)
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Volume 70 (1972)
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Volume 69 (1971)
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Volume 68 (1971)
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Volume 67 (1971)
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Volume 66 (1971)
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Volume 65 (1971)
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Volume 64 (1970)
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Volume 63 (1970)
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Volume 62 (1970)
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Volume 61 (1970)
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Volume 60 (1970)
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Volume 59 (1969)
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Volume 58 (1969)
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Volume 57 (1969)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)