- Volume 150, Issue 5, 2004
Volume 150, Issue 5, 2004
- Microbiology Comment
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- Physiological Adaptations In Amitochondriate Protists Of Clinical Significance
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tvcp12: a novel Trichomonas vaginalis cathepsin L-like cysteine proteinase-encoding gene
More LessTrichomonas vaginalis is the causative agent of trichomoniasis, one of the most common sexually transmitted diseases in humans. This protozoan has multiple proteinases that are mainly of the cysteine proteinase (CP) type, some of which are known to be involved in the parasite's virulence. Here, a novel T. vaginalis CP-encoding gene, tvcp12, was identified and characterized. tvcp12 is 948 bp long and encodes a predicted 34·4 kDa protein that has the characteristics of the papain-like CP family. TvCP12 does not appear to have a signal peptide, suggesting that this is a cytoplasmic CP. By Southern blot assays, the tvcp12 gene was found as a single copy in the T. vaginalis genome. Remarkably, Northern blot experiments showed a single transcript band of ∼1·3 kb in the mRNA obtained from parasites grown in low iron conditions and no transcript was observed in the mRNA from parasites grown in high iron conditions. By RT-PCR assays, a 270 bp band was amplified from the cDNA of parasites grown in low iron medium, which was very faint when cDNA from parasites grown in high iron conditions was used. Transcripts of the 3′ region obtained in both iron conditions presented differences in their poly(A) tail length. These data suggest that tvcp12 is another gene that is negatively regulated by iron and that the length of the poly(A) tail may be one of the factors involved in the iron-modulated protein expression.
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Production of ammonia by Tritrichomonas foetus and Trichomonas vaginalis
More LessProduction of ammonia is difficult to find among the various studies of amino acid metabolism in protozoa. Several studies suggest that catabolism of arginine to ammonium is important for the growth of trichomonads. Trichomonads are amitochondriate zooflagellates that thrive under microaerophilic and anaerobic conditions. The authors were able to detect accumulation of ammonium ions and ammonia in cultures of Tritrichomonas foetus and Trichomonas vaginalis, including those resistant to metronidazole. Ammonium ions and ammonia were detected using the indophenol colorimetric method. Cells incubated overnight under an ambient oxygen gas phase had 0·9 mM soluble ammonium (NH4 + and NH3) or a 20 % greater concentration of ammonium relative to sterile growth medium that had been incubated similarly. Production of ammonia itself was confirmed by analysis of a wick that was moistened with sulfuric acid (20 mM) and placed above the liquid in sealed cultures of a strain of Trichomonas vaginalis. The wicks from these cultures captured the equivalent of 0·048 mM volatile ammonia (NH3) from the liquid as compared to 0·021 mM volatile ammonia from sterile medium after overnight incubation. Intact trichomonads, 0·7×106 cells ml−1 equivalent to 0·7 mg protein ml−1, incubated in Doran's buffer with or without (1 mM) l-arginine produced significant amounts of soluble ammonium (0·07 mM and 0·04 mM, respectively) during 60 min. The results indicate that ammonium ions and the more irritating ammonia are significant metabolites of trichomonads. In addition, based upon end-product amounts, it appears that the rate of arginine metabolism is of the same order of magnitude as that for carbohydrate metabolism by trichomonads.
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PCR-based identification of zoonotic isolates of Blastocystis from mammals and birds
More LessThe genotype of Blastocystis isolated from humans and animals is highly polymorphic. Therefore, it is important to compare the genotypes of Blastocystis isolates from humans and animals to determine the zoonotic potential of animal isolates. PCR-based genotype classification using known sequence-tagged site (STS) primers allows identification of zoonotic isolates of animal origin. To this end, 51 isolates from monkeys, cattle, pigs, chickens, quails and pheasants were subjected to genotype analysis using seven kinds of STS primers. Out of the 51 isolates, 39 were identified as one of the known genotypes, four showed mixed genotypes, and eight were unknown genotypes as these were negative for all STS primers. When these results were combined with previous studies on 41 isolates from animals and compared with the diversity of genotypes of 102 human Blastocystis hominis isolates, 67·4 % (62/92) of isolates from mammals and birds were identical to human B. hominis genotypes. Since the unknown genotype of human origin had been placed into an additional clade in the small-subunit rRNA gene phylogeny, further molecular study on the eight isolates of unknown genotype from the present study will facilitate our understanding of their zoonotic potential.
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Phylogenetic identification of Pneumocystis murina sp. nov., a new species in laboratory mice
More LessPneumocystis is a fungal genus that contains multiple species. One member of the genus that has not been formally analysed for its phylogenetic relationships and possible species status is the Pneumocystis found in laboratory mice, Pneumocystis murina sp. nov. (type strain ATCC PRA-111T=CBS 114898T), formerly known as Pneumocystis carinii f. sp. muris. To advance research in this area, approximately 3000 bp of additional DNA sequence were obtained from the locus encoding rRNAs. This sequence and others were used to determine genetic distances between P. murina and other members of the genus. These distances indicated that P. murina DNA is most similar to that of the species of Pneumocystis found in laboratory rats. Nevertheless, P. murina is at least as diverged from these other Pneumocystis species as species in other fungal genera are from each other. The 18S rRNA gene sequence divergence exhibited by P. murina could not be ascribed to accelerated evolution of this gene as similar levels of divergence were observed at seven other loci. When five genes were used to construct phylogenetic trees for five Pneumocystis taxa, including P. murina, all the trees had the same topology, indicating that genes do not flow among these taxa. The gene trees were all strongly supported by statistical tests. When sequences from the rRNA-encoding locus were used to estimate the time of divergence of P. murina, the results indicated that P. murina is as old as the mouse. Taken together, these data support previous recognition of multiple species in the genus and indicate that P. murina is a phylogenetic species as well.
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Molecular typing of Pneumocystis jirovecii found in formalin-fixed paraffin-embedded lung tissue sections from sudden infant death victims
Previous studies have provided histological evidence of an association between primary Pneumocystis infection and sudden infant death syndrome (SIDS). The aim of this work was to determine the species of clustered Pneumocystis organisms found in formalin-fixed paraffin-embedded (FFPE) lung tissue sections from Chilean sudden infant death (SID) victims. This approach needed first to optimize a DNA extraction method from such histological sections. For that purpose, the QIAamp DNA Isolation from Paraffin-Embedded Tissue method (Qiagen) was first tested on FFPE lung tissue sections of immunosuppressed Wistar rats inoculated with rat-derived Pneumocystis. Successful DNA extraction was assessed by the amplification of a 346 bp fragment of the mitochondrial large subunit rRNA gene of the Pneumocystis species using a previously described PCR assay. PCR products were analysed by direct sequencing and sequences corresponding to Pneumocystis carinii were found in all the samples. This method was then applied to FFPE lung tissue sections from Chilean SID victims. Pneumocystis jirovecii was successfully identified in the three tested samples. In conclusion, an efficient protocol for isolating PCR-ready DNA from FFPE lung tissue sections was developed. It established that the Pneumocystis species found in the lungs of Chilean SID victims was P. jirovecii.
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Similar genotypes of Pneumocystis jirovecii in different forms of Pneumocystis infection
This study describes the genotyping of Pneumocystis jirovecii organisms isolated from three groups of patients that developed diverse forms of P. jirovecii infection; the patients were monitored in the same French hospital. Forty archival specimens from 13 adults with Pneumocystis pneumonia, eight adults colonized by P. jirovecii and 19 immunocompetent infants infected with the fungus contemporaneously with a bronchiolitis episode were analysed retrospectively. Genotyping was performed by analysis of sequences of the internal transcribed spacer (ITS)1 and ITS2 regions, and of the dihydropteroate synthase (DHPS) locus. At the ITS regions, a high diversity of genotypes, identical main genotypes (B1a3 and B2a1) and the occurrence of mixed infections (more than one genotype) were observed in the three patient groups. At the DHPS locus, the results indicated the presence of mutants in the two adult groups, as well as in the infant group. Consequently, at these two independent genomic regions, P. jirovecii isolates from patients who developed different forms of infection and who lived in the same geographical region presented common characteristics. These results suggest that patients infected with P. jirovecii, whatever the form of infection they present, are part of a common human reservoir for P. jirovecii.
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Inability of Pneumocystis organisms to incorporate bromodeoxyuridine suggests the absence of a salvage pathway for thymidine
More LessBecause thymidine metabolism is a potential target for therapy of Pneumocystis pneumonia, it was investigated whether Pneumocystis organisms have a salvage pathway for thymidine by administering 5-bromo-2′-deoxyuridine (BrdU) to mice and rats with Pneumocystis pneumonia. Although BrdU incorporation was detected in host cells, no incorporation was seen in Pneumocystis organisms infecting either rats or mice. This suggests that Pneumocystis organisms do not have a salvage pathway for thymidine, and that inhibitors of de novo synthesis, such as thymidylate synthase inhibitors, may be effective drugs for treating Pneumocystis pneumonia.
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The plasma membrane of microaerophilic protists: oxidative and nitrosative stress
The trans-plasma-membrane electrochemical potential of microaerophilic protists was monitored by the use of voltage-sensitive charged lipophilic fluorophores; of the many available probes, the anionic oxonol dye bis(1,3-dibarbituric acid)-trimethine oxonol [DiBAC4(3)] is an example of one which has been successfully employed using fluorescence microscopy, confocal laser-scanning microscopy and flow cytometry. Several microaerophilic protists have been investigated with this dye; these were Giardia intestinalis, Trichomonas vaginalis, Tritrichomonas foetus, Hexamita inflata and Mastigamoeba punctachora. Under conditions where they exhibit normal vitality, these organisms exclude DiBAC4(3) by virtue of their maintenance of a plasma-membrane potential (negative inside). Uptake of the fluorophore is indicative of disturbance to this membrane (i.e. by inhibition of pump/leak balance, blockage of channels or generation of ionic leaks), and is indicative of metabolic perturbation or environmental stress. Here, it is shown that oxidative or nitrosative stress depolarizes the plasma membranes of the aforementioned O2-sensitive organisms and allows DiBAC4(3) influx. Oxonol uptake thereby provides a sensitive and early indication of plasma-membrane perturbation by agents that may lead to cytotoxicity and eventually to cell death by necrotic or apoptotic pathways.
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Intron-containing β-tubulin transcripts in Cryptosporidium parvum cultured in vitro
More LessThe genome of Cryptosporidium parvum contains a relatively small number of introns, which includes the β-tubulin gene with only a single intron. Recently, it was observed that the intron was not removed from some of the β-tubulin transcripts in the late life cycle stages cultured in vitro. Although normally spliced β-tubulin mRNA was detected in all parasite intracellular stages by RT-PCR (e.g. HCT-8 or Caco-2 cells infected with C. parvum for 12–72 h), at 48–72 h post-infection unprocessed β-tubulin transcripts containing intact introns started to appear in parasite mRNA within infected host cells. The intron-containing transcripts could be detected by fluorescence in situ hybridization (FISH) using an intron-specific probe. The intron-containing β-tubulin transcripts appeared unique to the in vitro-cultured C. parvum, since they were not detected in parasite-infected calves at 72 h. As yet, it is unclear whether the late life cycle stages of C. parvum are partially deficient in intron-splicing or the intron-splicing processes have merely slowed, both of which would allow the detection of intron-containing transcripts. Another possible explanation is that the decay in transcript processing might simply be due to the onset of parasite death. Nonetheless, the appearance of intron-containing transcripts coincides with the arrest of C. parvum development in vitro. This unusual observation prompts speculation that the abnormal intron-splicing of β-tubulin transcripts may be one of the factors preventing complete development of this parasite in vitro. Furthermore, the presence of both processed and unprocessed introns in β-tubulin transcripts in vitro may provide a venue for studying overall mechanisms for intron-splicing in this parasite.
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Functional characterization of replication protein A2 (RPA2) from Cryptosporidium parvum
More LessReplication protein A (RPA) is a heterotrimeric complex of single-stranded DNA-binding proteins that play multiple roles in eukaryotic DNA metabolism. The RPA complex is typically composed of heterologous proteins (termed RPA1, RPA2 and RPA3) in animals, plants and fungi, which possess different functions. Previously, two distinct, short-type RPA large subunits (CpRPA1 and CpRPA1B) from the apicomplexan parasite Cryptosporidium parvum were characterized. Here are reported the identification and characterization of a putative middle RPA subunit (CpRPA2) from this unicellular organism. Although the CpRPA2 gene encodes a predicted 40·1 kDa peptide, which is larger than other RPA2 subunits characterized to date, Western blot analysis of oocyst preparations detected a native CpRPA2 protein with a molecular mass of approximately 32 kDa, suggesting that CpRPA2 might undergo post-translational cleavage or the gene was translated at an alternative start codon. Immunofluorescence microscopy using a rabbit anti-CpRPA2 antibody revealed that CpRPA2 protein was mainly distributed in the cytosol (rather than the nuclei) of C. parvum sporozoites. Semi-quantitative RT-PCR data indicated that CpRPA2 was differentially expressed in a tissue culture model with highest expression in intracellular parasites infecting HCT-8 cells for 36 and 60 h. Sequence comparison suggests that RPA2 is a group of poorly conserved proteins. Nonetheless, functional analyses of recombinant proteins confirmed that CpRPA2 is a single-stranded DNA-binding protein and that it could serve as an in vitro phosphorylation target by a DNA-dependent protein kinase. The minimal length of poly(dT) required for CpRPA2 binding is 17 nucleotides, and the DNA-binding capability was inhibited by phosphorylation in vitro. These observations provide additional evidence on the divergence of RPA proteins between C. parvum and host, implying that the parasite DNA replication machinery could be explored as a chemotherapeutic target.
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Differential expression and interaction of transcription co-activator MBF1 with TATA-binding protein (TBP) in the apicomplexan Cryptosporidium parvum
More LessAll gene-specific transcriptional activators initiate gene transcriptions by binding to promoter sequences and recruiting general transcription factors including TATA-binding protein (TBP) to upstream of targeted genes. Some of them require multiprotein bridging factors (MBFs); for example, the type 1 MBF (MBF1) which interconnects the gene activator with TBP. In this study, the properties of a previously cloned type 1 multiprotein bridging factor (CpMBF1) and a newly identified TBP (CpTBP1) from the apicomplexan Cryptosporidium parvum were investigated. Genes encoding both proteins were differentially expressed as determined by semi-quantitative RT-PCRs during the parasite life cycle, but in different patterns. The highest level of expression of CpMBF1 was in the well-developed intracellular parasites, whereas that of CpTBP1 was found in intact oocysts and late intracellular stages, possibly correlated with the formation of oocysts. Both CpMBF1 and CpTBP1 were expressed as maltose-binding protein fusion proteins. The function of CpTBP1 was confirmed by its ability to bind a biotinylated DNA oligonucleotide containing TATA consensus sequence. The interaction between CpMBF1 and CpTBP1 was also observed by an electrophoretic mobility shift assay. Since little is known about the regulation and control of gene activity in C. parvum, this study may point to a new direction for the study of gene activation associated with the development of the complex life cycle of this parasite.
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Polyamine metabolism in a member of the phylum Microspora (Encephalitozoon cuniculi): effects of polyamine analogues
The uptake, biosynthesis and catabolism of polyamines in the microsporidian parasite Encephalitozoon cuniculi are detailed with reference to the effects of oligoamine and arylamine analogues of polyamines. Enc. cuniculi, an intracellular parasite of mammalian cells, has both biosynthetic and catabolic enzymes of polyamine metabolism, as demonstrated in cell-free extracts of mature spores. The uptake of polyamines was measured in immature, pre-emergent spores isolated from host cells by Percoll gradient. Spermine was rapidly taken up and metabolized to spermidine and an unknown, possibly acetamidopropanal, by spermidine/spermine N 1-acetyltransferase (SSAT) and polyamine oxidase (PAO). Most of the spermidine and the unknown product were found in the cell incubation medium, indicating they were released from the cell. bis(Ethyl) oligoamine analogues of polyamines, such as SL-11144 and SL-11158, as well as arylamine analogues [BW-1, a bis(phenylbenzyl) 3-7-3 analogue] blocked uptake and interconversion of spermine at micromolar levels and, in the case of BW-1, acted as substrate for PAO. The Enc. cuniculi PAO activity differed from that found in mammalian cells with respect to pH optimum, substrate specificity and sensitivity to known PAO inhibitors. SL-11158 inhibited SSAT activity with a mixed type of inhibition in which the analogue had a 70-fold higher affinity for the enzyme than the natural substrate, spermine. The interest in Enc. cuniculi polyamine metabolism and the biochemical effects of these polyamine analogues is warranted since they cure model infections of Enc. cuniculi in mice and are potential candidates for human clinical trials.
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Amino sugar phosphate levels in Giardia change during cyst wall formation
More LessThe parasite Giardia intestinalis exists as a trophozoite (vegetative) that infects the human small intestine, and a cyst (infective) that is shed in host faeces. Cyst viability in the environment depends upon a protective cyst wall, which consists of proteins and a unique β(1-3) GalNAc homopolymer. UDP-GalNAc, the precursor for this polysaccharide, is synthesized from glucose by an enzyme pathway that involves amino sugar phosphate intermediates. Using a novel method of microanalysis by capillary electrophoresis, the levels of amino sugar phosphate intermediates in trophozoites before encystment, during a period of active encystment and after the peak of encystment were measured. These levels were used to deduce metabolic control of amino sugar phosphates associated with encystment. Levels of amino sugar phosphate intermediates increased during encystment, and then decreased to nearly non-encysting levels. The most pronounced increase was in glucosamine 6-phosphate, which is the first substrate unique in this pathway, and which is the positive effector for the pathway's putative rate-controlling enzyme, UDP-GlcNAc pyrophosphorylase. Moreover, more UDP-GalNAc than UDP-GlcNAc, its direct precursor, was detected at 24 h. It is postulated that the enhanced UDP-GalNAc is a result of enhanced synthesis of UDP-GlcNAc by the pyrophosphorylase, and its preferential conversion to UDP-GalNAc. These results suggest that kinetics of amino sugar phosphate synthesis in encysting Giardia favours the direction that supports cyst wall synthesis. The enzymes involved in synthesis of UDP-GalNAc and its conversion to cyst wall might be potential targets for therapeutic inhibitors of Giardia infection.
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Menadione kills trophozoites and cysts of Giardia intestinalis
Production of reactive oxygen species by redox cycling in the presence of low levels of oxygen has been studied as a possible approach to anti-protozoal chemotherapeutic strategy. Incubation of the diplomonad flagellate Giardia intestinalis with 2-methy-1,4-naphthoquinone (menadione), under anaerobic conditions, gave UV absorption changes characteristic of reduction to menadiol; partial reversal was observed on admitting O2. Under microaerobic conditions, similar to those on the surface of the jejunal mucosa, trophozoites consumed O2 rapidly in the presence of menadione; reaction products included singlet O2 (monitored by single photon counting of O2-dependent low-level chemiluminescence) and H2O2 (measured by the formation of Complex I of microperoxidase). Trophozoites became swollen and incapable of regulatory volume control; these irreversible responses led to loss of motility, cessation of flagellar activity and cell death. Comparison of the sensitivities of trophozoites to metronidazole and menadione gave LC50 values (μg ml−1) of 1·2 and 0·7, respectively; corresponding values for cysts (measured by in vitro excystation capacities) were >50 and 1·3. Menadione (LD50 in mice, 0·5 g kg−1) is therefore a potentially more useful and general anti-giardial agent than metronidazole, as it is active against cysts as well as trophozoites.
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Cyst wall synthase: N-acetylgalactosaminyltransferase activity is induced to form the novel N-acetylgalactosamine polysaccharide in the Giardia cyst wall
More LessUridine-5′-diphospho-N-acetylgalactosamine (UDP-GalNAc) is required in the formation of the outer filamentous wall of Giardia and is synthesized by inducible enzymes in the cytosol of encysting trophozoites. In this study, an inducible enzyme activity that is associated with a particle population isolated from encysting Giardia is reported, and this activity exclusively incorporates [1-14C]GalNAc (from UDP-[14C]GalNAc) into an ethanol precipitate with the same properties as the filamentous cyst wall of Giardia. This ethanol precipitate exhibits characteristics of Giardia cyst wall filaments in that both contain GalNAc as the only sugar moieties and are SDS-insoluble, proteinase- and alkali-resistant and acid-hydrolysable. However, since the precise chemical nature of the ethanol precipitate remains unknown, this enzyme activity is referred to tentatively as cyst wall synthase (CWS). CWS activity peaks in cells between 24 and 36 h of encystment and exhibits a high affinity and marked specificity for UDP-GalNAc as its substrate. UDP-N-acetylglucosamine, UDP-glucose, UDP-galactose, d-glucosamine and d-galactosamine were not incorporated into the ethanol precipitate. Partially purified CWS activity exhibits an apparent K m of 0·048 mM for UDP-GalNAc, a V max of 0·70 nmol min−1 (mg protein)−1 and a requirement for divalent cations in the following order of preference: Ca2+, Mg2+>Co2+>>>Mn2+, Zn2+. EDTA inhibits CWS activity.
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Mitosomes of Entamoeba histolytica are abundant mitochondrion-related remnant organelles that lack a detectable organellar genome
More LessThe existence of mitochondrion-related relict organelles (mitosomes) in the amitochondrial human pathogen Entamoeba histolytica and the detection of extranuclear DNA-containing cytoplasmic structures (EhKOs) has led to the suggestion that a remnant genome from the original mitochondrial endosymbiont might have been retained in this organism. This study reports on the mutually exclusive distribution of Cpn60 and extranuclear DNA in E. histolytica and on the distribution of Cpn60-containing mitosomes in this parasite. In situ nick-translation coupled to immunofluorescence microscopy failed to detect the presence of DNA in mitosomes, either in fixed parasite trophozoites or in partially purified organellar fractions. These results indicate that a remnant organellar genome has not been retained in E. histolytica mitosomes and demonstrate unequivocally that EhKOs and mitosomes are distinct and unrelated cellular structures.
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Expression in fibroblasts and in live animals of Entamoeba histolytica polypeptides EhCP112 and EhADH112
EhCPADH is an immunogenic, heterodimeric protein that is formed by EhCP112 (cysteine protease) and EhADH112 (adhesin), polypeptides involved in Entamoeba histolytica's cytopathic effect, target-cell adherence and phagocytosis. The EhCPADH complex is located in the plasma membrane and cytoplasmic vacuoles. Here, the independent expression of EhCP112 and EhADH112 in fibroblasts and hamsters was analysed. Also investigated was the immunological response in animals independently inoculated with plasmid pcDNA-Ehcp112, which carries the complete cysteine protease-encoding gene, or with plasmid pcDNA-Ehadh112, which carries the C terminus of the adhesin-encoding gene, or with a mixture of both. Both proteins were expressed in the plasma membranes of the transfected fibroblasts. EhCP112 was toxic for the mammalian cells. Proteins were also independently expressed in hamsters after inoculation with the plasmids. Their expression was indirectly evaluated by the presence of antibodies in the inoculated animals. Remarkably, co-immunization of the animals with the two DNA plasmids resulted in an earlier and higher anti-E. histolytica IgG induction than immunization with separate plasmids. In contrast, the cellular immune response was not noticeably improved by the plasmid mixture. Interestingly, protection against liver abscesses was detected only in animals that received the plasmid mixture and no protection was observed in hamsters independently inoculated with plasmid pcDNA-Ehcp112 or pcDNA-Ehadh112.
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Spore morphotypes of Thelohania solenopsae (microsporidia) described microscopically and confirmed by PCR of individual spores microdissected from smears by position ablative laser microbeam microscopy
More LessDevelopment of Thelohania solenopsae, a parasite of the red imported fire ant (Solenopsis invicta), until recently was thought to include formation of two types of spores: unicellular meiospores, maturing inside sporophorous vesicles in sets of eight (octospores); and Nosema-like binuclear free spores. Megaspores, discovered in 2001, develop primarily in alates and are morphologically distinct from the two previously known types of spores. The role of megaspores in the T. solenopsae life cycle, as well as their existence, has been questioned. The current research includes light and electron microscopic descriptions of the three major spore morphotypes characteristic of T. solenopsae development. In addition, individual octospores and megaspores were isolated into groups of 8–20 from methanol-fixed and Calcofluor-stained smears of the infected ants for subsequent PCR analysis by the laser pressure catapulting function of a position ablative laser microbeam microscope, a technique applied for the first time to research of microsporidia. The PCR-amplified SSU rDNA nucleotide sequences from octospores and megaspores were identical. This, along with the consistency with which megaspores are detected in infected ants, demonstrates that megaspores are integral to the life cycle of T. solenopsae.
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- Cell And Developmental Biology
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Enhanced motility of a Proteus mirabilis strain expressing hybrid FlaAB flagella
More LessProteus mirabilis has two tandemly arranged flagellin-encoding genes, flaA and flaB. flaA is transcribed from a σ 28 promoter, while flaB is silent. flaA and flaB can undergo reversible rearrangement to produce a set of hybrid genes referred to as flaAB. Flagellins composed of FlaAB protein have a different amino acid sequence and are antigenically distinct from flagellin composed of FlaA, implicating flagellin gene conversion as a putative virulence mechanism for P. mirabilis. The change in amino acid sequence is also hypothesized to alter the filament helix and, hence, affect the motility of FlaAB-expressing strains. To test this hypothesis, the motility of wild-type P. mirabilis was compared with that of a strain, DF1003, locked into the FlaAB+ hybrid phase, under conditions of altered ionic strength, pH and viscosity. Cell motion tracking analysis showed that DF1003 has wild-type swimming velocity at physiological conditions, but moves significantly faster and travels further compared to the wild-type at NaCl concentrations greater than 170 mM. DF1003 is also significantly faster than the wild-type at pH 5·2, 5·8 and 8·2, and at 5 and 10 % polyvinylpyrrolidone. Measurements of amplitude and wavelength for isolated flagella subjected to pH 5·8 or 425 mM NaCl showed a loss of helical structure in FlaA flagella compared to FlaAB filaments, a feature that could significantly affect motility under these conditions. These results support a hypothesis that FlaAB flagellin imparts a motile advantage to P. mirabilis in conditions that otherwise may impede bacterial movement. In a broader context, flagellar antigenic variation, commonly thought to serve as means to avoid host defences, may also enhance motility in other bacterial species, thus aiding in the adaptation and survival of the cells.
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- Biochemistry And Molecular Biology
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The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression
More LessThe 60 nucleotides encoding the signal peptide of the Neisseria meningitidis membrane-bound lytic transglycosylase (MltA) homologue GNA33 were found to exert a negative regulatory effect on expression of GNA33 from either a T7- or a Plac-driven system in Escherichia coli. Down-regulation was observed to occur at the transcriptional/post-transcriptional level and could possibly be ascribed to the formation of a stem–loop secondary structure within the signal peptide sequence. Slowing down the transcription rate through inhibition/titration of the RNA polymerase resulted in a considerable increase in mRNA accumulation, suggesting that a better coupling of translation to transcription would impede the formation of the putative secondary structure. Screening of synonymous mutations in the signal peptide sequence that showed high-level expression of an in-frame fusion to a reporter resulted in the isolation of several deletion mutants lacking most of the sequence participating in the putative secondary structure. Interestingly, the increase in the steady-state mRNA level observed in deletion mutants was higher, reaching a 300-fold increment, than that found in substitution mutants. Our results support the hypothesis that the rate of transcription controls the formation of a secondary structure in the region of the GNA33 transcript corresponding to the signal peptide sequence and this, when formed, negatively regulates expression.
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Unique organization and regulation of the mrx fimbrial operon in Xenorhabdus nematophila
More LessXenorhabdus nematophila, a Gram-negative bacterium belonging to the Proteus clade of the family Enterobacteriaceae, forms a mutualistic association with the soil nematode Steinernema carpocapsae. The nematode invades insects and releases Xenorhabdus into the haemolymph, where it participates in insect killing. To begin to understand the role of fimbriae in the unique life cycle of Xenorhabdus, the organization and expression of the mrx fimbrial operon was analysed. The mrx operon contained only five structural genes (mrxACDGH), making it one of the smallest chaperone-usher fimbrial operons studied to date. Unlike the mrp operon of Proteus mirabilis, a site-specific recombinase was not linked to the mrx operon. The intergenic region between the major fimbrial gene (mrxA) and the usher gene (mrxC) lacked a mrpB-like gene, but contained three tandem inverted repeat sequences located downstream of mrxA. A 940 nt mrxA-containing mRNA was the major transcript produced in cells growing on agar, while an mrx polycistronic mRNA was produced at low levels. A canonical σ 70 promoter, identified upstream of mrxA, was not subject to promoter inversion. Fimbriae were not produced in an lrp-mutant strain, suggesting that the leucine-responsive regulatory protein, Lrp, plays a role in the regulation of the mrx operon. These findings show that the genetic organization and regulation of the mrx operon is in several respects distinct from other chaperone-usher fimbrial operons.
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The Fur-like protein Mur of Rhizobium leguminosarum is a Mn2+-responsive transcriptional regulator
More LessIn wild-type Rhizobium leguminosarum, the sitABCD operon specifies a Mn2+ transporter whose expression is severely reduced in cells grown in the presence of this metal. Mutations in the R. leguminosarum gene, mur (manganese uptake regulator), whose product resembles the Fur transcriptional regulator, cause high-level expression of sitABCD in the presence of Mn2+. In gel-shift mobility assays, purified R. leguminosarum Mur protein bound to at least two regions near the sitABCD promoter region, although this DNA has no conventional consensus Fur-binding sequences (fur boxes). Thus, in contrast to γ-proteobacteria, where Fur binds Fe2+, the R. leguminosarum Fur homologue, Mur, act as a Mn2-responsive transcriptional regulator.
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Increased transcription rates correlate with increased reversion rates in leuB and argH Escherichia coli auxotrophs
More LessEscherichia coli auxotrophs of leuB and argH were examined to determine if higher rates of transcription in derepressed genes were correlated with increased reversion rates. Rates of leuB and argH mRNA synthesis were determined using half-lives and concentrations, during exponential growth and at several time points during 30 min of amino acid starvation. Changes in mRNA concentration were primarily due to increased mRNA synthesis and not to increased stability. Four strains of E. coli amino acid auxotrophs, isogenic except for relA and argR, were examined. In both the leuB and argH genes, rates of transcription and mutation were compared. In general, strains able to activate transcription with guanosine tetraphosphate (ppGpp) had higher rates of mRNA synthesis and mutation than those lacking ppGpp (relA2 mutants). argR knockout strains were constructed in relA + and relA mutant strains, and rates of both argH reversion and mRNA synthesis were significantly higher in the argR knockouts than in the regulated strains. A statistically significant linear correlation between increased rates of transcription and mutation was found for data from both genes. In general, changes in mRNA half-lives were less than threefold, whereas changes in rates of mRNA synthesis were often two orders of magnitude. The results suggest that specific starvation conditions target the biosynthetic genes for derepression and increased rates of transcription and mutation.
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Role of homoserine and threonine pathway intermediates as precursors for the biosynthesis of aminoethoxyvinylglycine in Streptomyces sp. NRRL 5331
More LessThe genes hom, thrB and thrC, encoding homoserine dehydrogenase, homoserine kinase (HK) and threonine synthase, respectively, involved in the last steps of threonine biosynthesis, have been studied in Streptomyces sp. NRRL 5331, the producer of the ethylene synthetase inhibitor aminoethoxyvinylglycine (AVG), in order to determine their role in the biosynthesis of AVG. Different null mutants were obtained by plasmid-mediated disruption of each of the three genes. thrC gene disruption had no effect on AVG production, while the disruption of thrB blocked HK activity and substantially reduced the yield of this metabolite, probably due to the accumulation of homoserine and/or methionine which have a negative effect on AVG biosynthesis. Disruption of hom (thrA) completely blocked AVG biosynthesis, indicating that homoserine lies at the branching point of the aspartic-acid-derived biosynthetic route that leads to AVG. The four carbon atoms of the vinylglycine moiety of AVG derive, therefore, from homoserine.
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Properties of haemolysin E (HlyE) from a pathogenic Escherichia coli avian isolate and studies of HlyE export
Haemolysin E (HlyE) is a novel pore-forming toxin first identified in Escherichia coli K-12. Analysis of the 3-D structure of HlyE led to the proposal that a unique hydrophobic β-hairpin structure (the β-tongue, residues 177–203) interacts with the lipid bilayer in target membranes. In seeming contradiction to this, the hlyE sequence from a pathogenic E. coli strain (JM4660) that lacks all other haemolysins has been reported to encode an Arg residue at position 188 that was difficult to reconcile with the proposed role of the β-tongue. Here it is shown that the JM4660 hlyE sequence encodes Gly, not Arg, at position 188 and that substitution of Gly188 by Arg in E. coli K-12 HlyE abolishes activity, emphasizing the importance of the head domain in HlyE function. Nevertheless, 76 other amino acid substitutions were confirmed compared to the HlyE protein of E. coli K-12. The JM4660 HlyE protein was dimeric, suggesting a mechanism for improving toxin solubility, and it lysed red blood cells from many species by forming 36–41 Å diameter pores. However, the haemolytic phenotype of JM4660 was found to be unstable due to defects in HlyE export, indicating that export of active HlyE is not an intrinsic property of the protein but requires additional components. TnphoA mutagenesis of hlyE shows that secretion from the cytoplasm to the periplasm does not require the carboxyl-terminal region of HlyE. Finally, disruption of genes associated with cell envelope function, including tatC, impairs HlyE export, indicating that outer membrane integrity is important for effective HlyE secretion.
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- Biodiversity And Evolution
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NifH and NifD phylogenies: an evolutionary basis for understanding nitrogen fixation capabilities of methanotrophic bacteria
More LessThe ability to utilize dinitrogen as a nitrogen source is an important phenotypic trait in most currently known methanotrophic bacteria (MB). This trait is especially important for acidophilic MB, which inhabit acidic oligotrophic environments, highly depleted in available nitrogen compounds. Phylogenetically, acidophilic MB are most closely related to heterotrophic dinitrogen-fixing bacteria of the genus Beijerinckia. To further explore the phylogenetic linkage between these metabolically different organisms, the sequences of nifH and nifD gene fragments from acidophilic MB of the genera Methylocella and Methylocapsa, and from representatives of Beijerinckia, were determined. For reference, nifH and nifD sequences were also obtained from some type II MB of the alphaproteobacterial Methylosinus/Methylocystis group and from gammaproteobacterial type I MB. The trees constructed for the inferred amino acid sequences of nifH and nifD were highly congruent. The phylogenetic relationships among MB in the NifH and NifD trees also agreed well with the corresponding 16S rRNA-based phylogeny, except for two distinctive features. First, different methods used for phylogenetic analysis grouped the NifH and NifD sequences of strains of the gammaproteobacterial MB Methylococcus capsulatus within a clade mainly characterized by Alphaproteobacteria, including acidophilic MB and type II MB of the Methylosinus/Methylocystis group. From this and other genomic data from Methylococcus capsulatus Bath, it is proposed that an ancient event of lateral gene transfer was responsible for this aberrant branching. Second, the identity values of NifH and NifD sequences between Methylocapsa acidiphila B2 and representatives of Beijerinckia were clearly higher (98·5 and 96·6 %, respectively) than would be expected from their 16S rRNA-based relationships. Possibly, these two bacteria originated from a common acidophilic dinitrogen-fixing ancestor, and were subject to similar evolutionary pressure with regard to nitrogen acquisition. This interpretation is corroborated by the observation that, in contrast to most other diazotrophs, M. acidiphila B2 and Beijerinckia spp. are capable of active growth on nitrogen-free media under fully aerobic conditions.
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Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients
More LessType IV pili (TFP) are important colonization factors of the opportunistic pathogen Pseudomonas aeruginosa, involved in biofilm formation and attachment to host cells. This study undertook a comprehensive analysis of TFP alleles in more than 290 environmental, clinical, rectal and cystic fibrosis (CF) isolates of P. aeruginosa. Based on the results, a new system of nomenclature is proposed, in which P. aeruginosa TFP are divided into five distinct phylogenetic groups. Each pilin allele is stringently associated with characteristic, distinct accessory genes that allow the identification of the allele by specific PCR. The invariant association of the pilin and accessory genes implies horizontal transfer of the entire locus. Analysis of pilin allele distribution among isolates from various sources revealed a striking bias in the prevalence of isolates with group I pilin genes from CF compared with non-CF human sources (P<0·0001), suggesting this particular pilin type, which can be post-translationally modified by glycosylation via the action of TfpO (PilO), may confer a colonization or persistence advantage in the CF host. This allele was also predominant in paediatric CF isolates (29 of 43; 67·4 %), showing that this bias is apparent early in colonization. Group I pilins were also the most common type found in environmental isolates tested. To the authors' knowledge, this is the first example of a P. aeruginosa virulence factor allele that is strongly associated with CF isolates.
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Emergence of phenotypic variants upon mismatch repair disruption in Pseudomonas aeruginosa
MutS is part of the bacterial mismatch repair system that corrects point mutations and small insertions/deletions that fail to be proof-read by DNA polymerase activity. In this work it is shown that the disruption of the P. aeruginosa mutS gene generates the emergence of diverse colony morphologies in contrast with its parental wild-type strain that displayed monomorphic colonies. Interestingly, two of the mutS morphotypes emerged at a high frequency and in a reproducible way and were selected for subsequent characterization. One of them displayed a nearly wild-type morphology while the other notably showed, compared with the wild-type strain, increased production of pyocyanin and pyoverdin, lower excretion of LasB protease and novel motility characteristics, mainly related to swarming. Furthermore, it was reproducibly observed that, after prolonged incubation in liquid culture, the pigmented variant consistently emerged from the mutS wild-type-like variant displaying a reproducible event. It is also shown that these P. aeruginosa mutS morphotypes not only displayed an increase in the frequency of antibiotic-resistant mutants, as described for clinical P. aeruginosa mutator isolates, but also generated mutants whose antibiotic-resistant levels were higher than those measured from spontaneous resistant mutants derived from wild-type cells. It was also found that both morphotypes showed a decreased cytotoxic capacity compared to the wild-type strain, leading to the emergence of invasive variants. By using mutated versions of a tetracycline resistance gene, the mutS mutant showed a 70-fold increase in the reversion frequency of a +1 frameshift mutation with respect to its parental wild-type strain, allowing the suggestion that the phenotypical diversity generated in the mutS population could be produced in part by frameshift mutations. Finally, since morphotypical diversification has also been described in clinical isolates, the possibility that this mutS diversification was related to the high frequency hypermutability observed in P. aeruginosa CF isolates is discussed.
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- Environmental Microbiology
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Effects of high light on transcripts of stress-associated genes for the cyanobacteria Synechocystis sp. PCC 6803 and Prochlorococcus MED4 and MIT9313
More LessCyanobacteria constitute an ancient, diverse and ecologically important bacterial group. The responses of these organisms to light and nutrient conditions are finely controlled, enabling the cells to survive a range of environmental conditions. In particular, it is important to understand how cyanobacteria acclimate to the absorption of excess excitation energy and how stress-associated transcripts accumulate following transfer of cells from low- to high-intensity light. In this study, quantitative RT-PCR was used to monitor changes in levels of transcripts encoding chaperones and stress-associated proteases in three cyanobacterial strains that inhabit different ecological niches: the freshwater strain Synechocystis sp. PCC 6803, the marine high-light-adapted strain Prochlorococcus MED4 and the marine low-light-adapted strain Prochlorococcus MIT9313. Levels of transcripts encoding stress-associated proteins were very sensitive to changes in light intensity in all of these organisms, although there were significant differences in the degree and kinetics of transcript accumulation. A specific set of genes that seemed to be associated with high-light adaptation (groEL/groES, dnaK2, dnaJ3, clpB1 and clpP1) could be targeted for more detailed studies in the future. Furthermore, the strongest responses were observed in Prochlorococcus MED4, a strain characteristic of the open ocean surface layer, where hsp genes could play a critical role in cell survival.
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Influences of temperature, salinity and starvation on the motility and chemotactic response of Vibrio anguillarum
More LessThe role of growth factors for the motility and chemotaxis of the fish pathogen Vibrio anguillarum was determined. Cells of V. anguillarum were chemotactic to serine in the temperature range 5–25 °C and in 0·8–2·7 % NaCl. The chemotactic response was significantly higher at 25 °C than at 5 or 15 °C. Growth in medium with 1·5 % NaCl gave a higher response than growth with 3 % NaCl; when the salinity of the chemotaxis buffer was raised, the chemotactic response was reduced. The role of starvation was also studied; V. anguillarum showed a high chemotactic response after starvation for 2 and 8 days. Motility and chemotaxis are important virulence factors for this bacterium. Not only was the ability to perform chemotactic motility maintained after starvation, but also it was shown that starvation does not interfere with the ability of the organism to cause infection in rainbow trout after a bath challenge. The swimming speed was reduced at lower temperatures. Within the range of salinity and starvation studied, the motile cells swam with the same velocity, indicating that V. anguillarum under all the examined conditions has a functional flagellum and rotates it with constant speed. Phenamil, a specific inhibitor of Na+-driven flagella, reduced the motility of both starved and non-starved cells of V. anguillarum indicating that, in both cases, a Na+ motive force drives the flagellum.
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- Genes And Genomes
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Two relA/spoT homologous genes are involved in the morphological and physiological differentiation of Streptomyces clavuligerus
This study is focused on the involvement of the unusual nucleotide (p)ppGpp during the morphological and physiological differentiation of Streptomyces clavuligerus. In particular, the functional and structural elements of two genes encoding the proteins RelA and Rsh were identified. The relA gene encodes an 843 aa protein (RelA), while the rsh gene encodes a 738 aa protein (Rsh). The relA and rsh genes were disrupted by the insertion of a hygromycin resistance gene and an apramycin resistance gene, respectively. The synthesis of ppGpp in the relA gene-disrupted mutant was completely eliminated under conditions of starvation for amino acids, whereas synthesis persisted, but was greatly reduced in the rsh gene-disrupted mutant. The relA gene-disrupted mutant had a bald appearance on agar plate cultures and retarded growth in submerged culture, while the rsh-disrupted mutant was unchanged in growth characteristics relative to the wild-type culture. The production of both clavulanic acid and cephamycin C were completely abolished in the relA-disrupted mutant. Thus, it is concluded that the relA gene rather than rsh is essential for morphological and physiological differentiation in S. clavuligerus and that RelA primarily governs the stringent response of S. clavuligerus to starvation for amino acids.
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Linear versus circular mitochondrial genomes: intraspecies variability of mitochondrial genome architecture in Candida parapsilosis
More LessThe yeast species Candida parapsilosis, an opportunistic pathogen, exhibits genetic and genomic heterogeneity. To assess the polymorphism at the level of mitochondrial DNA (mtDNA), the organization of the mitochondrial genome in strains belonging to the three variant groups of this species was investigated. Although these analyses revealed a group-specific restriction fragment pattern of mtDNA, strains belonging to different groups appear to have similar genes in the same gene order. An extensive survey of C. parapsilosis isolates uncovered surprising alterations in the molecular architecture of their mitochondrial genome. A screening strategy for strains harbouring mtDNA with rearranged architecture showed that nearly all strains from groups I and III possess linear mtDNA molecules terminating with arrays of tandem repeat units, while most of the group II strains have a circular mitochondrial genome. In addition, it was found that linear genophores in mitochondria of strains from different groups differ in the sequence of the mitochondrial telomeric repeat unit. The occurrence of altered forms of mtDNA among C. parapsilosis strains opens up the unique possibility to address questions concerning the evolutionary origin and replication strategy of linear and circular genomes in mitochondria.
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Global analysis of gene expression in an rpoN mutant of Listeria monocytogenes
The role of the alternative σ 54 factor, encoded by the rpoN gene, was investigated in Listeria monocytogenes by comparing the global gene expression of the wild-type EGDe strain and an rpoN mutant. Gene expression, using whole-genome macroarrays, and protein content, using two-dimensional gel electrophoresis, were analysed. Seventy-seven genes and nine proteins, whose expression was modulated in the rpoN mutant as compared to the wild-type strain, were identified. Most of the modifications were related to carbohydrate metabolism and in particular to pyruvate metabolism. However, under the conditions studied, only the mptACD operon was shown to be directly controlled by σ 54. Therefore, the remaining modifications seem to be due to indirect effects. In parallel, an in silico analysis suggests that σ 54 may directly control the expression of four different phosphotransferase system (PTS) operons, including mptACD. PTS activity is known to have a direct effect on the pyruvate pool and on catabolite regulation. These results suggest that σ 54 is mainly involved in the control of carbohydrate metabolism in L. monocytogenes via direct regulation of PTS activity, alteration of the pyruvate pool and modulation of carbon catabolite regulation.
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Comparison of the structure–activity relationships of the integron-associated recombination sites attI3 and attI1 reveals common features
More LessIncorporation of gene cassettes into integrons occurs by IntI-mediated site-specific recombination between a 59-base element (59-be) site in the cassette and an attI site in the integron. While the 59-be sites share common features and are recognized by several different IntI recombinases, the sequences of attI sites are not obviously related and are preferentially recognized by the cognate IntI. To determine the features of attI sites that are required for recombination proficiency, the structure–activity relationships of a second attI site, the attI3 site from the class 3 integron, were examined. The attI3 site was confined to within a region consisting of 68 bp from the integron backbone and 15 bp from the adjacent cassette. This region includes four IntI3-binding sites, as assessed by gel shift and methylation interference studies. Two of the binding sites are inversely oriented and constitute a simple site that includes the recombination crossover point. The two additional binding sites appear to be directly oriented and one of them is essential for efficient recombination of the attI3 site with a 59-be, but not for recombination with a second full-length attI3 site, which occurs at 100-fold lower frequency. The fourth site enhances attI3 with 59-be recombination 10-fold. The finding that the organization and overall properties of attI3 are very similar to those of attI1 indicates that these features are likely to be common to all attI sites.
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- Pathogens And Pathogenicity
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Molecular and functional characterization of type I signal peptidase from Legionella pneumophila
Legionella pneumophila is a facultative intracellular Gram-negative rod-shaped bacterium that has become an important cause of both community-acquired and nosocomial pneumonia. Numerous studies concerning the unravelling of the virulence mechanism of this important pathogen have been initiated. As evidence is now accumulating for the involvement of protein secretion systems in bacterial virulence in general, the type I signal peptidase (LepB) of L. pneumophila was of particular interest. This endopeptidase plays an essential role in the processing of preproteins carrying a typical amino-terminal signal peptide, upon translocation across the cytoplasmic membrane. This paper reports the cloning and the transcriptional analysis of the L. pneumophila lepB gene encoding the type I signal peptidase (SPase). Reverse transcription PCR experiments showed clear lepB expression when L. pneumophila was grown both in culture medium, and also intracellularly in Acanthamoeba castellanii, a natural eukaryotic host of L. pneumophila. In addition, LepB was shown to be encoded by a polycistronic mRNA transcript together with two other proteins, i.e. a LepA homologue and a ribonuclease III homologue. SPase activity of the LepB protein was demonstrated by in vivo complementation analysis in a temperature-sensitive Escherichia coli lepB mutant. Protein sequence and predicted membrane topology were compared to those of leader peptidases of other Gram-negative human pathogens. Most strikingly, a strictly conserved methionine residue in the substrate binding pocket was replaced by a leucine residue, which might influence substrate recognition. Finally it was shown by in vivo experiments that L. pneumophila LepB is a target for (5S,6S)-6-[(R)-acetoxyethyl]-penem-3-carboxylate, a specific inhibitor of type I SPases.
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Induction of Mycobacterium avium growth restriction and inhibition of phagosome–endosome interactions during macrophage activation and apoptosis induction by picolinic acid plus IFNγ
More LessTreatment of mouse macrophages with picolinic acid (PA) and γ-interferon (IFNγ) led to the restriction of Mycobacterium avium proliferation concomitant with the sequential acquisition of metabolic changes typical of apoptosis, mitochondrial depolarization, annexin V staining and caspase activation, over a period of up to 5 days. However, triggering of cell death by ATP, staurosporine or H2O2 failed to affect mycobacterial viability. In contrast to untreated macrophages where extensive interactions between phagosomes and endosomes were observed, phagosomes from treated macrophages lost the ability to acquire endosomal dextran. N-Acetylcysteine was able to revert both the anti-mycobacterial activity of treated macrophages as well as the block in phagosome–endosome interactions. The treatment, however, induced only a minor increase in the acquisition of lysosomal markers, namely Lamp-1, and did not increase to any great extent the acidification of the phagosomes. These data thus suggest that the anti-mycobacterial activity of PA and IFNγ depends on the interruption of intracellular vesicular trafficking, namely the blocking of acquisition of endosomal material by the microbe.
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Genome structure in the vole bacillus, Mycobacterium microti, a member of the Mycobacterium tuberculosis complex with a low virulence for humans
Mycobacterium microti, a member of the Mycobacterium tuberculosis complex, is phylogenetically closely related to M. tuberculosis, differing in a few biochemical properties. However, these species have different levels of virulence in different hosts; most notably M. microti shows lower virulence for humans than M. tuberculosis. This report presents genomic comparisons using DNA microarray analysis for an extensive study of the diversity of M. microti strains. Compared to M. tuberculosis H37Rv, 13 deletions were identified in 12 strains of M. microti, including the regions RD1 to RD10, which are also missing in Mycobacterium bovis BCG. In addition, four new deleted regions, named MiD1, RD1β, MiD2 and MiD3, were identified. DNA sequencing was used to define the extent of most of the deletions in one strain. Although RD1 of M. bovis BCG and M. microti is thought to be crucial for attenuation, in this study, three of the four M. microti strains that were isolated from immunocompetent patients had the RD1 deletion. In fact, only the RD3 deletion was present in all of the strains examined, although deletions RD7, RD8 and MiD1 were found in almost all the M. microti strains. These deletions might therefore have some relation to the different host range of M. microti. It was also noticeable that of the 12 strains studied, only three were identical; these strains were all isolated from immunocompetent humans, suggesting that they could have arisen from a single source. Thus, this study shows that it is difficult to ascribe virulence to any particular pattern of deletion in M. microti.
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Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes
Clostridium botulinum type C 16S progenitor toxin consists of a neurotoxin (NTX), a non-toxic non-HA (NTNH), and a haemagglutinin (HA). The HA acts as an adhesin, allowing the 16S toxin to bind to intestinal epithelial cells and erythrocytes. In type C, these bindings are dependent on sialic acid. The HA consists of four distinct subcomponents designated HA1, HA2, HA3a and HA3b. To identify the binding subcomponent(s) of HA of type C 16S toxin, all of the HA-subcomponents and some of their precursor forms were produced as recombinant proteins fused to glutathione S-transferase (GST). These proteins were evaluated for their capacity to adhere to intestinal epithelial cells of guinea pig and human erythrocytes. GST-HA1, GST-HA3b and GST-HA3 (a precursor form of HA3a and HA3b) bound intestinal epithelial cells and erythrocytes, whereas GST alone, GST-HA2 and GST-HA3a did not. GST-HA3b and GST-HA3 showed neuraminidase-sensitive binding to the intestinal epithelial cells and erythrocytes, whereas GST-HA1 showed neuraminidase-insensitive binding. TLC binding assay revealed that GST-HA3b and GST-HA3 recognized sialosylparagloboside (SPG) and GM3 in the ganglioside fraction of the erythrocytes, like native type C 16S toxin [ Inoue, K. et al. (1999). Microbiology 145, 2533–2542 ]. On the other hand, GST-HA1 recognized paragloboside (PG; an asialo- derivative of SPG) in addition to SPG and GM3. Deletion mutant analyses of GST-HA3b showed that the C-terminal region of HA3b is important for its binding activity. Based on these data, it is concluded that the HA component contains two distinct carbohydrate-binding subcomponents, HA1 and HA3b, which recognize carbohydrates in different specificities.
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Evolution of multi-resistance plasmids in Australian clinical isolates of Escherichia coli
More LessPlasmids allow the movement of genetic material, including antimicrobial resistance genes, between bacterial species and genera. They frequently mediate resistance to multiple antimicrobials and can result in the acquisition by a pathogen of resistance to all or most clinically relevant antimicrobials. Unfortunately, there are still large gaps in our understanding of how new multi-resistance plasmids evolve. Five Australian clinical institutions collaborated in this study of multi-resistance plasmids in clinical isolates of Escherichia coli. We characterized 72 resistance plasmids in terms of the antimicrobial resistance profile they conferred, their size and their incompatibility group. Restriction fragment length polymorphisms were used to determine the genetic relationships between the plasmids. Relationships between the host cells were determined using multi-locus enzyme electrophoresis. A lack of correlation between the evolutionary history of the host cells and their plasmids suggests that the horizontal transfer of resistance plasmids between strains of E. coli is common. The resistance plasmids were very diverse, with a wide range of resistance profiles and a lack of discrete evolutionary lineages. Multi-resistance plasmids did not evolve via the co-integrative capture of smaller resistance plasmids; rather, the roles of recombination and the horizontal movement of mobile genetic elements appeared to be most important.
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Cryptococcus neoformans Ilv2p confers resistance to sulfometuron methyl and is required for survival at 37 °C and in vivo
More LessAcetolactate synthase catalyses the first common step in isoleucine and valine biosynthesis and is the target of several classes of inhibitors. The Cryptococcus neoformans ILV2 gene, encoding acetolactate synthase, was identified by complementation of a Saccharomyces cerevisiae ilv2 mutant. C. neoformans is highly resistant to the commercially available acetolactate synthase inhibitor, sulfometuron methyl (SM). Expression of C. neoformans ILV2 in S. cerevisiae conferred SM resistance, indicating that the SM resistance of C. neoformans is due, at least in part, to C. neoformans Ilv2p. The C. neoformans ILV2 gene was disrupted. The ilv2 mutants were auxotrophic for isoleucine and valine and the auxotrophy was satisfied by these amino acids only when proline, and not ammonium, was the nitrogen source, indicating nitrogen regulation of amino acid transport. ilv2 mutants rapidly lost viability at 37 °C and when starved for isoleucine and valine. Consistent with these phenotypes, an ilv2 mutant was avirulent and unable to survive in mice. Because C. neoformans Ilv2p is required for virulence and survival in vivo, inhibitors of branched-chain amino acid biosynthesis may make valuable antifungal agents.
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SpeB modulates fibronectin-dependent internalization of Streptococcus pyogenes by efficient proteolysis of cell-wall-anchored protein F1
More LessSpeB is a cysteine proteinase and virulence determinant secreted by the important human pathogen Streptococcus pyogenes. Recent investigations have suggested a role for SpeB in streptococcal entry into human cells. However, conflicting data concerning the contribution of SpeB to internalization have been presented. Protein F1 is a cell-wall-attached fibronectin (Fn)-binding protein that is present in a majority of streptococcal isolates and is important for internalization. This study shows that protein F1 is efficiently degraded by SpeB, and that removal of protein F1 from the bacterial surface leads to reduced internalization. Whereas M1 protein and protein H, two additional surface proteins of S. pyogenes that bind human plasma proteins, are protected from proteolytic degradation by their ligands, protein F1 is readily cleaved by SpeB also when in complex with Fn. This finding, and the connection between the presence of Fn at the bacterial surface and entry into human cells, suggest that SpeB plays a role in the regulation of the internalization process.
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- Physiology
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Stress-responsive proteins are upregulated in Streptococcus mutans during acid tolerance
More LessStreptococcus mutans is an important pathogen in the initiation of dental caries as the bacterium remains metabolically active when the environment becomes acidic. The mechanisms underlying this ability to survive and proliferate at low pH remain an area of intense investigation. Differential two-dimensional electrophoretic proteome analysis of S. mutans grown at steady state in continuous culture at pH 7·0 or pH 5·0 enabled the resolution of 199 cellular and extracellular protein spots with altered levels of expression. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry identified 167 of these protein spots. Sixty-one were associated with stress-responsive pathways involved in DNA replication, transcription, translation, protein folding and proteolysis. The 61 protein spots represented isoforms or cleavage products of 30 different proteins, of which 25 were either upregulated or uniquely expressed during acid-tolerant growth at pH 5·0. Among the unique and upregulated proteins were five that have not been previously identified as being associated with acid tolerance in S. mutans and/or which have not been studied in any detail in oral streptococci. These were the single-stranded DNA-binding protein, Ssb, the transcription elongation factor, GreA, the RNA exonuclease, polyribonucleotide nucleotidyltransferase (PnpA), and two proteinases, the ATP-binding subunit, ClpL, of the Clp family of proteinases and a proteinase encoded by the pep gene family with properties similar to the dipeptidase, PepD, of Lactobacillus helveticus. The identification of these and other differentially expressed proteins associated with an acid-tolerant-growth phenotype provides new information on targets for mutagenic studies that will allow the future assessment of their physiological significance in the survival and proliferation of S. mutans in low pH environments.
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Proteome analysis of Streptococcus mutans metabolic phenotype during acid tolerance
More LessTwo-dimensional gel electrophoretic analysis of the proteome of Streptococcus mutans grown at a steady state in a glucose-limited anaerobic continuous culture revealed a number of proteins that were differentially expressed when the growth pH was lowered from pH 7·0 to pH 5·0. Changes in the expression of metabolic proteins were generally limited to three biochemical pathways: glycolysis, alternative acid production and branched-chain amino acid biosynthesis. The relative level of expression of protein spots representing all of the enzymes associated with the Embden–Meyerhof–Parnas pathway, and all but one of the enzymes involved in the major alternative acid fermentation pathways of S. mutans, was identified and measured. Proteome data, in conjunction with end-product and cell-yield analyses, were consistent with a phenotypic change that allowed S. mutans to proliferate at low pH by expending energy to extrude excess H+ from the cell, while minimizing the detrimental effects that result from the uncoupling of carbon flux from catabolism and the consequent imbalance in NADH and pyruvate production. The changes in enzyme levels were consistent with a reduction in the formation of the strongest acid, formic acid, which was a consequence of the diversion of pyruvate to both lactate and branched-chain amino acid production when S. mutans was cultivated in an acidic environment.
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Functional analysis of the transcriptional activator XlnR from Aspergillus niger
The transcriptional activator XlnR from Aspergillus niger is a zinc binuclear cluster transcription factor that belongs to the GAL4 superfamily. Several putative structural domains in XlnR were predicted using database and protein sequence analysis. Thus far, only the functionality of the N-terminal DNA-binding domain has been determined experimentally. Deletion mutants of the xlnR gene were constructed to localize the functional regions of the protein. The results showed that a putative C-terminal coiled-coil region is involved in nuclear import of XlnR. After deletion of the C-terminus, including the coiled-coil region, XlnR was found in the cytoplasm, while deletion of the C-terminus downstream of the coiled-coil region resulted in nuclear import of XlnR. The latter mutant also showed increased xylanase activity, indicating the presence of a region with an inhibitory function in XlnR-controlled transcription. Previous findings had already shown that a mutation in the XlnR C-terminal region resulted in transcription of the structural genes under non-inducing conditions. A regulatory model of XlnR is presented in which the C-terminus responds to repressing signals, resulting in an inactive state of the protein.
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Volumes and issues
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Volume 170 (2024)
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Volume 106 (1978)
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Volume 104 (1978)
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Volume 96 (1976)
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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)