- Volume 145, Issue 8, 1999
Volume 145, Issue 8, 1999
- Review Article
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- Antigens And Immunity
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A chimaeric plant virus vaccine protects mice against a bacterial infection
The plant virus cowpea mosaic virus (CPMV) is an efficient carrier of foreign peptides for the generation of strong humoral immune responses. Peptides derived from both viruses and bacteria are strongly immunogenic when displayed on the surface of CPMV and elicit high titres of peptide-specific antibody. However, the protective effects of antibodies generated using bacterial epitopes in this system have yet to be demonstrated. In this study the ability of chimaeric virus particles (CVPs) to afford protection against bacterial infection was assessed. Immunization of outbred mice with CPMV expressing a peptide derived from outer-membrane protein F of Pseudomonas aeruginosa (CPMV-PAE5) generated high titres of P. aeruginosa-specific IgG that opsonized the bacteria for phagocytosis by human neutrophils and afforded protection upon challenge with two different immunotypes of P. aeruginosa in a model of chronic pulmonary infection. When examined 8 d after challenge, CVP-immunized mice had fewer severe lung lesions and fewer bacteria in their lungs compared to mice immunized with wild-type virus. Different levels of protection were seen with CPMV-PAE5 when Freund's or alum adjuvants were used. These studies highlight the ability of CVPs to generate protective immunity against infectious disease agents.
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Antigenic variants in Bordetella pertussis strains isolated from vaccinated and unvaccinated children
Bordetella pertussis shows polymorphism in two proteins, pertactin (Prn) and the pertussis toxin (PT) S1 subunit, which are important for immunity. A previous study has shown antigenic shifts in these proteins in the Dutch B. pertussis population, and it was suggested that these shifts were driven by vaccination. The recent Italian clinical trial provided the opportunity to compare the frequencies of Prn and PT S1 subunit variants in strains isolated from unvaccinated children, and from children vaccinated with two acellular and one whole-cell pertussis vaccine. Four Prn variants (Prn1, Prn2, Prn3 and Prn5) were found in the 129 strains analysed. Prn1, Prn2 and Prn3 have been described previously, whereas Prn5 is a novel variant. Prn1, Prn2, Prn3 and Prn5 were found in, respectively, 6, 41, 51 and 2% of the strains. The B. pertussis strains used to produce the vaccines administered in the clinical trial were found to produce Prn1, or a type which differed from Prn1 in one amino acid. The frequency of the Prn1 variant was found to be lowest in the strains isolated from vaccinated groups, suggesting that Prn1 strains are more affected by vaccine-induced immunity than Prn2 and Prn3 strains. Only one PT S1 type (S1A) was observed in the examined strains, which was distinct from the types produced by the vaccine strains (S1B and S1D). The S1A type also predominates in the Dutch B. pertussis population. The genetic relationship among B. pertussis strains analysed by IS1002-based DNA fingerprinting revealed that three fingerprint types predominate, representing more than 70% of the strains. Prn2 strains showed a greater variety of fingerprint types compared to Prn3, suggesting that Prn3 has emerged more recently. The results are discussed in the light of vaccine-driven evolution.
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Use of a primate model system to identify Chlamydia trachomatis protein antigens recognized uniquely in the context of infection
More LessA primate model system was used to identify Chlamydia trachomatis antigens uniquely recognized in the context of infection. Serum antibody titres were measured in cynomolgus monkeys challenged urethrally with C. trachomatis serovar L2 elementary bodies (EBs). High-titre sera from these primates were used, in parallel with antisera against killed C. trachomatis EBs, to differentially screen an expression library of C. trachomatis serovar L2 DNA. Four clones were recognized only by antisera from infected monkeys. Sequence analysis revealed that three of these immunoreactive clones overlap a common ORF, designated ORF D242 (encoding p242), in the C. trachomatis genome database. The fourth clone contains two complete ORFs, each encoding 32 kDa proteins that share identity with Treponema pallidum TroA and TroB (ORFs D067 and D068 in the C. trachomatis database, respectively). lmmunoblot analysis of Escherichia coli lysates expressing C. trachomatis TroA, TroB and p242 fusion proteins showed that p242 and TroA, but not TroB, were detected by the sera collected from infected primates. Antibodies directed at TroA and p242 were also detected in sera from several C. trachomatis-infected patients, demonstrating that these proteins are also recognized by humans following infection. Immunoblot analysis with antibody against TroA and p242 also demonstrated that both antigens are present in higher abundance in infected ChoK1 cells relative to purified C. trachomatis EBs. Immunofluorescence microscopy shows that TroA and p242 are both localized to intracellular developmental forms at the margins of growing inclusions. Collectively, these studies identify two C. trachomatis proteins that are under-represented in EBs and are recognized uniquely in the context of infection.
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Mycoplasma synoviae surface protein MSPB as a recombinant antigen in an indirect ELISA
Mycoplasma synoviae is a poultry pathogen causing respiratory disease and synovitis. A number of serological assays have been developed for diagnosis of M. synoviae infection; however, they lack sensitivity and/or are prone to false-positive reactions. Using a combination of PCR and expression cloning, four overlapping regions (regions 1-4) of the surface antigen MSPB of M. synoviae WVU-1853 were expressed in a bacterial expression system.
Immunostaining of the resultant polypeptides with chicken sera raised against different M. synoviae strains, or Mycoplasma gallisepticum S6, suggested that region 4 contained a highly antigenic and species-specific domain (amino acids 178-213) of MSPB. A fusion protein of region 4 was expressed in the pMAL expression system and purified from cold-osmotic-shock fluids of Escherichia coli cells for use in an indirect ELISA. The potential of the purified antigen for detection of M. synoviae antibodies was assessed with sera obtained from chickens experimentally infected with different strains of M. synoviae or M. gallisepticum, or from uninoculated chickens. All the sera from M. synoviae-inoculated chickens provided higher absorbance values than those from M. gallisepticum-inoculated or uninoculated chickens. Chickens inoculated with M. synoviae 86079/7NS had detectable increases of serum anti-MSPB immunoglobulins at day 7 after inoculation. These studies have identified the most antigenic region of one of the major species-specific surface proteins of M. synoviae, and shown the potential of this protein as a serodiagnostic reagent.
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- Biochemistry
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The role of malic enzyme in the regulation of lipid accumulation in filamentous fungi
More LessThe hypothesis is advanced that NADP+-malic enzyme (ME; EC 1.1.1.40) is an important activity in regulating the extent of lipid accumulation in filamentous fungi. In Mucor circinelloides, a fungus capable of accumulating only 25% (w/w, dry wt) lipid, even under the most propitious conditions, ME disappears 15-20 h after nitrogen exhaustion, coincident with the cessation of lipid accumulation. In contrast, ME in Mortierella alpina, a fungus capable of accumulating 50% (w/w, dry wt) lipid, remains active for over 60 h after N-exhaustion during which time lipid accumulation continues. No other enzyme activity studied, including the lipogenic enzymes acetyl-CoA carboxylase, fatty acid synthase, diacyglycerol acyltransferase, ATP: citrate lyase and the NADPH-generating enzymes glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP+: isocitrate dehydrogenase, demonstrated any correlation with the accumulation of storage lipid in either fungus. Full activity of ME is restored in Mr. circinelloides within 4 h by adding NH+ 4 to the cultures, but this is prevented by adding cycloheximide as an inhibitor of protein synthesis. This suggests that the decrease in ME activity occurs due to down-regulation of the ME gene.
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Cryptocandin, a potent antimycotic from the endophytic fungus Cryptosporiopsis cf. quercina
A unique lipopeptide antimycotic, termed cryptocandin, is described from Cryptosporiopsis cf. quercina, an endophytic fungus. Cryptocandin, with a molecular mass of 1079 Da, contains equimolar amounts of 3,4-dihydroxyhomotyrosine, 4-hydroxyproline, threonine, glutamine, 3-hydroxy-4-hydroxymethylproline, 4,5-dihydroxyornithine and palmitic acid. Cryptocandin is chemically related to well-known antimycotics, the echinocandins and pneumocandins, which are produced by such fungi as Zalerion arboricola, Pezicula spp. and Aspergillus spp. Cryptocandin has minimal inhibitory concentration values of 0-03-0-07 μg ml-1 against isolates of Candida albicans, Trichophyton mentagrophytes and Trichophyton rubrum. Cryptocandin is also active against a number of plant-pathogenic fungi including Sclerotinia sclerotiorum and Botrytis cinerea.
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- Development And Structure
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High cAMP in spores of Dictyostelium discoideum: association with spore dormancy and inhibition of germination
Signalling mechanisms involving cAMP have a well-documented role in the coordination of multicellular development and differentiation leading to spore formation in the social amoeba, Dictyostelium discoideum. The involvement of cAMP in the poorly understood developmental stages of spore dormancy and germination have been investigated in this study. Dormant spores contained up to 11-fold more cAMP than nascent amoebae. The spore cAMP levels were not constant, but typically underwent a surge at 14-18 d when spores acquired the ability to germinate spontaneously. The high cAMP levels decreased only during successful spore germination, i.e. emergence of nascent amoebae. The temporal pattern of cAMP decrease was complex and unique to the method of spore activation, supporting our hypothesis that exogenously (e.g. heat) activated and autoactivated spores germinate by different mechanisms. During heat-induced activation, transcription of acg (a gene encoding adenylyl cyclase associated with germination) correlated well with spore cAMP content. Young wild-type spores, incapable of spontaneous germination, maintained a uniformly high cAMP level, and spore cAMP levels also remained high if germination was inhibited. When activated spores were deactivated by applying increased osmotic pressure, cAMP concentrations rose and ultimately levelled off at the high levels typical of dormant spores. The correlation between high cAMP and failure to germinate was also evident when autoactivation was inhibited by the cAMP analogue, 8-bromo-cAMP. Also, spores from a strain (HTY217) with unrestrained protein kinase A activity were incapable of spontaneous germination. Overall, our experiments provide evidence for continued cAMP signalling in spores up to 18 d after sporulation and for linkages between elevated cAMP, spore deactivation and inhibition of spontaneous germination.
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Ammonium phosphate in sori of Dictyostelium discoideum promotes spore dormancy through stimulation of the osmosensor ACG
More LessThe sori of Dictyostelium discoideum (strains SG1, SG2, NC4 and V12) contained more than 100 mM ammonium phosphate. Glutamine synthetase (GS), which could remove ammonia from the sorus, was not present in 2-d-old dormant spores but enzyme activity returned to vegetative levels after spore germination. Based on mRNA blotting, the activity of this enzyme in germinating spores appeared to be transcriptionally controlled. At the same time that GS activity was increasing, ammonia was released from germinating spores. Exogenous ammonium ions at a concentration of 28 mM did not block germination nor modulate GS activity in nascent amoebae. It was concluded that the transcription and translation of GS is not environmentally regulated but is an integral part of the germination process, preparing nascent amoebae for vegetative growth. An exogenous concentration of 69 mM ammonium phosphate could maintain dormancy in spores of strains SG1 and SG2 for at least a week in the absence of any other inhibitory component from the sori. The inhibition was reversible at any time either by dilution or by washing the spores free of the ammonium ion. Spores of strain acg - were not inhibited by 100 mM ammonium phosphate. A model is presented in which GS in prespore cells serves as a sink for ammonia to allow the osmotically sensitive adenylyl cyclase aggregation protein (ACA) to activate protein kinase A (PKA) to induce fruiting-body formation. After fruiting-body formation is complete, the decline in GS and ACA activities in developing spores is offset by their replacement with the osmotically and ammonia-stimulated adenylyl cyclase osmosensor for germination (ACG). Ammonia and discadenine may act as separate signals to synergistically activate PKA by stimulating ACG activity while inhibiting cAMP phosphodiestrase activity in fully dormant spores.
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Membrane vesicles derived from Pseudomonas aeruginosa and Shigella flexneri can be integrated into the surfaces of other Gram-negative bacteria
More LessIncubation of intact Salmonella typhi Ty21a, Salmonella enterica serovar Typhimurium (Salmonella typhimurium) aroA or Escherichia coli DH5α with membrane vesicles (MVs) derived from either Shigella flexneri M90T or Pseudomonas aeruginosa dsp89 resulted in a significant incorporation of vesicle antigens into the outer membrane of the bacteria; each recipient strain possessed a surface mosaic of new Shigella and Pseudomonas antigens intermixed with the native antigens of the Salmonella or Escherichia strains. Electron microscopy of preparations during the integration of vesicle antigens revealed that the MVs rapidly fused with the outer membrane of the host strains. Western blot analysis of host bacteria confirmed the integration of foreign antigens. Quantitative analysis for binding and fusion of antigens using an ELISA showed that approximately 78·7 ± 12·8 ng of the Pseudomonas and 67·5 ± 13·8 ng of the Shigella LPSs (μg host protein)−1 were integrated into the Sal. typhimurium strain. Similar integrations of the Shigella or Pseudomonas vesicles were found with the E. coli or Sal. typhi strains. There was no loss of viability in the recipient bacteria after incorporation of the MV's, although vesicle antigens became diluted during continued growth as daughter cells shared the vesicle antigens. The new antigens were highly stable after being incorporated into recipient strains, being able to withstand storage of several months at 4°C as well as several cycles of freezing and thawing. Since the recipient bacteria are common vaccine strains, the procedure described here offers a simple efficient means of introducing exogenous surface antigens, in their native form, into the outer membranes of Gram-negative bacteria for possible vaccine use.
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- Environmental Microbiology
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A simplified subtractive hybridization protocol used to isolate DNA sequences specific to Xylella fastidiosa
More LessA simplified protocol of subtractive hybridization based on the technique of L. M. Kunkel, A. P. Monaco, W. Middlesworth, H. D. Ochs & S. A. Latt (1985, Proc Natl Acad Sci USA, 82, 4778-4782) was used to obtain DNA sequences specific to Xylella fastidiosa isolated from diseased citrus plants. As a driver, DNA extracted from bacteria showing different degrees of relatedness was used: Xy. fastidiosa 788 isolated from another host (plum), Xanthomonas campestris pv. campestris and Burkholderia gladioli strains. A DNA fragment, f14, showing no hybridization to the driver DNA, was used as a probe specific to Xy. fastidiosa from citrus and oleander. This fragment was sequenced and the predicted protein showed 40% similarity to the central region of flagellin of Escherichia coli serotypes H1 and H12. A pair of internal primers (f14-1 and f14-2) was designed for amplification of Xy. fastidiosa DNA. These primers detected Xy. fastidiosa strains isolated from citrus and oleander and yielded an amplification product of about 600 bp. They were also able to detect the bacteria in extracts from citrus plants with or without symptoms of disease. No amplification reaction was obtained using DNA extracted from other species and pathovars of Xanthomonas, Pseudomonas cichorii, Erwinia carotovora, Agrobacterium tumefaciens and phytopathogens of citrus (Xanthomonas axonopodis pv. citri) and coffee (Burkholderia andropogonis, P. cichorii, Pseudomonas syringae pv. garcae). The isolation of a DNA fragment specific to Xy. fastidiosa from citrus showed that the simplified protocol of subtractive hybridization used in this work is potentially applicable to other microorganisms.
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Identification of novel bacterial lineages as active members of microbial populations in a freshwater sediment using a rapid RNA extraction procedure and RT-PCR
More LessA rapid method for the extraction of RNA from the indigenous bacterial communities in environmental samples was developed. The method was tested using anoxic sediment samples from a productive freshwater lake (Priest Pot, Cumbria, UK). The simple protocol yielded rRNA and mRNA of a purity suitable for amplification by reverse transcriptase PCR (RT-PCR). The integrity of the RT-PCR was demonstrated by amplifying 165 rRNA and mRNA for the mercury resistance regulatory gene merR. The diversity of 16S rRNA sequences recovered from RNA and DNA extracted from anoxic Priest Pot sediments was analysed. The 5′ end of extracted 165 rRNA was amplified by RT-PCR and the 165 rRNA PCR products were cloned and sequenced to identify active constituents of the sediment bacterial community. Corresponding analyses were performed upon DNA templates from the same sediment samples. Partial 16S rRNA sequences were obtained from a total of 147 clones (71 rRNA-derived and 76 rDNA-derived). The clone libraries included sequences related to Pirellula staleyi, an aerobic planktonic member of the Planctomycetales, and the recently described candidate bacterial division OP5. Sequences from these groups were recovered in libraries generated from a DNA template but were also present in RNA-derived libraries. Previous studies of anoxic environments have identified sequences most closely related to Pirellula spp. This study, which utilized RT-PCR of 165 rRNA, has provided the first evidence that Pirellula-like bacteria are active in situ in an anoxic environment. Furthermore, members of the recently described candidate division, OP5, were also identified as active constituents of the bacterial community of anoxic Priest Pot sediments. This not only supports the widespread occurrence of OP5 members in diverse environments but suggests that they are active under anoxic conditions.
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- Genetics And Molecular Biology
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The glyceraldehyde-3-phosphate dehydrogenase of Clostridium acetobutylicum: isolation and purification of the enzyme, and sequencing and localization of the gap gene within a cluster of other glycolytic genes
More LessGlyceraldehyde-3-phosphate dehydrogenase was purified from Clostridium acetobutylicum by sequential ammonium sulfate precipitation, gel filtration and anion-exchange chromatography (to a specific activity of 27 U mg-1). The enzyme had a molecular mass of 40 kDa as determined by SDS-PAGE and a native molecular mass of 160 kDa as determined by nondenaturing PAGE, indicating that it has a homotetrameric composition. Its pH optimum was between 8.5 and 9.3. The corresponding gene (gap) was cloned and sequenced from C. acetobutylicum DSM 792 and found to cluster with other genes of enzymes from the glycolytic pathway (pgk, phosphoglycerate kinase; tpi, triosephosphate isomerase; pgm(i), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase). No sequences resembling rho-independent transcription terminators were found in the intergenic regions. A plasmid carrying the clostridial gap gene complemented an Escherichia coli gap mutant.
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Regulated interactions between partner and non-partner sensors and response regulators that control glycopeptide resistance gene expression in enterococci
More LessTranscription of the vanA and vanB glycopeptide resistance gene clusters is regulated by the VanRS and VanRBSB two-component regulatory systems, respectively. Histidine to glutamine substitutions were introduced at positions 164 of VanS and 233 of VanSB to prevent autophosphorylation of the sensor kinases and transfer of the phosphate groups to the VanR and VanRB response regulators. VanSH164Q and VanSBH233Q abolished activation of VanR and VanRB by host kinases. The phosphatase activity of VanSBH233Q was negatively modulated by vancomycin whereas VanSH164Q prevented transcription of the resistance genes under all growth conditions. Cross-talk was detected between VanRB and VanS in a vanSB null mutant. VanR is required for activation of promoters PR and PH allowing transcription of the regulatory (vanRS) and resistance (vanHAXYZ) genes, respectively. Under non-inducing conditions, activation of VanR by cross-talk was blocked by the presence of a multicopy plasmid carrying PH . Presence of the high-affinity VanR-binding sites of the regulatory region of PH on the multicopy vector probably sequestered VanR, thereby preventing autoactivation of the PR promoter. Under such circumstances, stimulation of the host kinase by glycopeptides or moenomycin was required for expression of the resistance genes.
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Genetic and physiological studies of the CiaH-CiaR two-component signal-transducing system involved in cefotaxime resistance and competence of Streptococcus pneumoniae
More LessA mutation in the ciaH gene of Streptococcus pneumoniae induces cefotaxime resistance and transformation deficiency. ciaH encodes a putative sensor protein that belongs to the family of signal-transducing histidine kinases. This gene is adjacent to ciaR, which encodes a DNA-binding protein involved in the regulation of genes responding to environmental signals sensed by the histidine kinase. The authors have characterized a mutation that induces reversion of both cefotaxime resistance and transformation deficiency. It is a T/A deletion in the ciaR gene resulting in the synthesis of a truncated protein containing only 125 amino acids instead of 224. The ciaH mutation requires a functional CiaR protein for expression. Northern blot analysis, using ciaR-ciaH as a probe, revealed one mRNA from the wild-type strain, indicating that the two genes constitute an operon. Comparisons of Northern blots show that the operon is constitutively activated in the strain carrying only the ciaH mutation. In the wild-type strain the activation occurs when the Ca2+ concentration is very low, demonstrating that Ca2+ is the environmental signal. The pleiotropic effects caused by the ciaH mutation include sensitivity to antibiotics and toxins, the ability to form protoplasts and the susceptibility to lysis with deoxycholate. Null-mutants were constructed in both genes and the particular features of the ciaR null mutant determined. It is able to grow in choline-deprived medium, and competence development occurs in a phosphate-deprived competence medium (CH-maleate), suggesting that the CiaH-CiaR system regulates several pathways, including teiochoic acid synthesis.
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Involvement of the N- and C-terminal domains of Mycobacterium tuberculosis KatG in the protection of mutant Escherichia coli against DNA-damaging agents
More LessThe Mycobacterium tuberculosis KatG enzyme, like most hydroperoxidase I (HPI)-type catalases, consists of two related domains, each with strong similarity to the yeast cytochrome c peroxidase. The catalase-peroxidase activity is associated with the amino-terminal domain but currently no definite function has been assigned to the carboxy-terminal domain, although it may play a role in substrate binding. This paper reports another possible function of the KatG protein involving protection of the host cell against DNA-damaging agents. The M. tuberculosis katG gene, the 5′ domain and the 3′ domain were cloned separately, in-frame with the maltose-binding protein, into the vector pMAL-c2. These constructs were introduced into four DNA-repair mutants of Escherichia coli, DK1 (recA), AB1884 (uvrC), AB1885 (uvrB) and AB1886 (uvrA), which were then tested for their ability to survive treatment with UV light (254 nm), hydrogen peroxide (1·6 mg ml-1) and mitomycin C (6 μg ml-1). All three constructs conferred resistance to UV upon the recA E. coli cells, whereas resistance to mitomycin C was found in all repair mutants tested. Protection against hydrogen peroxide damage was less pronounced and predominantly found in the recA host. These results indicated that the M. tuberculosis katG gene can enhance DNA repair in E. coli, and that the 5′ and 3′ domains can function separately. UV sensitivity tests on Mycobacterium intracellulare and M. tuberculosis strains mutant in katG revealed that the katG gene product does not play an additive role in the survival of mycobacterial cells after exposure to short-wavelength UV irradiation, in repair-competent cells.
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Serotype 14 variants of the Spanish penicillin-resistant serotype 9V clone of Streptococcus pneumoniae arose by large recombinational replacements of the cpsA-pbp1a region
More LessThe high prevalence of penicillin resistance among Streptococcus pneumoniae isolates from Uruguay has been associated with the emergence of a penicillin-resistant clone of serotype 14. Isolates of this clone were identical by multilocus sequence typing to members of the Spanish penicillin-resistant serotype 9V clone and possessed indistinguishable forms of the penicillin-binding protein 2b and 2x genes. Their pbp1a genes were also identical, except at the 3′ end. The serotype 14 isolates were shown to be a variant of the Spanish serotype 9V clone which arose by a 22·2 kb recombinational replacement that introduced the capsular biosynthetic locus, and part of the neighbouring pbp1a gene, from a serotype 14 isolate. One end of the recombinational replacement was within the first gene of the capsular polysaccharide operon, cpsA, as the sequence of the upstream dexB gene, through most of cpsA, was identical in the penicillin-resistant serotype 9V and 14 isolates, but the sequences differed in the rest of cpsA and in cpsB. The other recombinational junction was at the end of the divergently transcribed pbp1a gene, which is approximately 11·6 kb downstream of the capsular biosynthetic locus. Isolates of this serotype variant were also detected in Spain and Denmark. Penicillin-resistant serotype 14 isolates from Poland were also closely related to the penicillin-resistant serotype 9V clone, but have emerged independently, as one end of the recombinational replacement was upstream of dexB and the other was within pbp1a, but at a different position from that in the serotype 14 variants from Uruguay, Spain and Denmark. Serotype 14 variants of the Spanish serotype 9V clone have therefore arisen on more than one occasion by large recombinational replacements that extend from the start of the cps region into the pbp1a gene.
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A gene family in Mycoplasma imitans closely related to the pMGA family of Mycoplasma gallisepticum
More LessThe avian pathogen Mycoplasma gallisepticum possesses a large gene family encoding lipoproteins which function as haemagglutinins. Representative species of the pneumoniae phylogenetic group of mycoplasmas were examined for the presence of genes homologous to members of this multigene family. Antisera against the pMGA1.1 lipoprotein recognized a 35 kDa protein in Mycoplasma imitans, but did not recognize proteins of Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma pirum, Mycoplasma penetrans or Mycoplasma iowae in Western blots. A fragment of the pMGA1.2 gene and oligonucleotide probes complementary to highly conserved coding and non-coding regions of pMGA genes bound to fragments of genomic DNA of M. imitans, but not to the genomes of M. genitalium, M. pneumoniae, M. pirum or M. penetrans, and only one probe bound to a fragment of the M. iowae genome. One homologue of the pMGA genes was amplified from the M. imitans genome by PCR and used as a probe to clone a 3.1 kbp DNA fragment from a library of HindIII-digested M. imitans genomic DNA. The contiguous DNA sequence of the PCR and HindIII clones was predicted to encode one complete and one partial ORF which shared some peptide sequence identity with the pMGA genes, including the signal peptidase II cleavage site and the proline-rich amino-terminal region. Like the pMGA genes, the M. imitans genes were found to be members of a large gene family, with an association with GAA trinucleotide repeats, a feature which distinguishes these two families from the homologous vlhA gene family in Mycoplasma synoviae. The identification of these gene families in three phylogenetically distinct avian mycoplasma species, but not in human mycoplasmas, suggests their horizontal transfer between species infecting the same host.
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HmsT, a protein essential for expression of the haemin storage (Hms+) phenotype of Yersinia pestis
More LessThe haemin storage (Hms) phenotype of Yersinia pestis has been shown to be involved in the blockage of fleas that is required for the transmission of plague from fleas to mammals. Previously, an operon encoding four genes, hmsHFRS, that are essential for the temperature-regulated Hms+ phenotype has been characterized. Here the isolation and characterization of a fifth gene, hmsT, that is essential for this phenotype is described. Conceptual translation of hmsT suggests it encodes a 44.8 kDa protein with a pl of 7.75. The gene for HmsT is located outside of the ~ 102 kb pgm locus of Y. pestis that contains the hmsHFRS operon. Hybridization studies indicate that Yersinia pseudotuberculosis but not Yersinia enterocolitica or Escherichia coli possesses a highly homologous gene. HmsT belongs to a family of PleD-related proteins with four highly conserved regions of homology. Although PleD is a regulator, the functions of the other members of this family have not been experimentally determined. The iron-responsive regulator, Fur, has previously been implicated in temperature regulation of the Hms phenotype. A good potential Fur-binding site (FBS) is located upstream of hmsT. Y. pestis M23 and two of five Y. pseudotuberculosis strains, which all exhibit a temperature-constitutive Hms phenotype, contain a 6 bp insertion in the putative FBS. E. coli MG1655 contains homologues of hmsHFRST (ycdSRQPT) but has an Hms- phenotype. Only ycdQ and ycdP complement mutations in their respective homologues, hmsR and hmsS, in Y. pestis.
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On-line monitoring of gene expression
More LessGene expression in cultures of Escherichia coli has been determined in situ and on-line by the use of an electrochemical sensor. Intact bacteria were used to monitor the induction of the lacZ gene; the onset of stationary phase was also monitored, using a reporter gene fused to the RpoS-dependent promoter of the osmY gene. The technique described can in principle be used to determine the activity of any promoter, with a variety of reporter genes. This technology is non-intrusive, allows real-time monitoring of gene expression, and will be useful in the study of growth regulation and development.
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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)