Heme binding by a bacterial repressor protein, the gene product of the ferric uptake regulation (fur) gene of Escherichia coli

The fur gene product, Fur, of Escherichia coli is a repressor when it binds Fe(II). Since heme and iron metabolism are closely linked and Fur is rich in histidine, a ligand for heme, the binding of heme to Fur was investigated. The oxidized Fur-heme complex is stable and low spin with a Soret maximum at 404 nm and no 620-nm band. CO coordinates with the reduced heme-Fur complex, causing a shift from 412 nm to 410 nm, and stabilizes it, increasing the half-life from 5 to 15 min. Circular dichroism (CD) spectra in the Soret region show heme bound in an asymmetric environment in Fur, both in the oxidized and reduced-CO forms. Quenching of tyrosine fluorescence by heme revealed rapid, tight binding (Kd < 1 microM) with an unusual stoichiometry of 1 heme:1 Fur dimer. Fur binds Mn(II), a model ligand for the endogenous Fe(II), much more weakly (Kd > 80 microM). Far-ultraviolet CD spectroscopy showed that the alpha-helix content of apo-Fur decreases slightly with heme binding, but increases with Mn(II) binding. Competition experiments indicated that heme interacts with Fur dimers at the same site as Mn(II) and can displace the metal. In contrast to Mn(II), Zn(II) did not quench the tyrosine fluoroescence of Fur, affected the CD spectrum less than Mn(II), but did bind in a manner which prevented heme from binding. In sum, Fur not only binds heme and Zn(II) with sufficient affinity to be biologically relevant, but the interactions that occur between these ligands and their effects on Mn(II) binding need to be taken into account when addressing the biological function of Fur.     

A putative adhesin gene cloned from Campylobacter jejuni

Thirteen Campylobacter jejuni strains of human origin showed differing behaviours when analysed for their ability to bind the Caco-2 cell line in vitro, suggesting variations in genetic complements and/or regulation. We designed an oligonucleotide probe corresponding to a highly conserved part of adhesins from various Gram-negative bacteria. Among our laboratory collection, Southern hybridization has demonstrated that only a discrete number of strains harbour this sequence. The corresponding gene has been cloned from our prototype strain and sequence analysis has confirmed homology with Gram-negative bacterial adhesins. The ORF corresponded to 869 amino acids; we named this protein P95. Protein sequence similarity assessment demonstrated that this gene product belongs to the family of proteins including the filamentous haemagglutinin of Bordetella pertussis and the high-molecular-weight surface-exposed adhesins of Haemophilus influenzae. Comparison of adhesion and hybridization results emphasized the involvement of this gene in an essential pathogenic process of Campylobacter.     

Differential regulation of chordin expression domains in mutant zebrafish

Patterning along the dorsal-ventral (D-V) axis of Xenopus and Drosophila embryos is believed to occur through a conserved molecular mechanism, with homologous proteins Chordin and Short gastrulation (Sog) antagonizing signaling by bone morphogenetic protein 4 (BMP-4) and Decapentaplegic (Dpp), respectively. We have isolated a zebrafish gene that is highly homologous to chordin and sog within cysteine-rich domains and exhibits conserved aspects of expression and function. As in Xenopus embryos, zebrafish chordin is expressed in the organizer region and transiently in axial mesoderm. Injection of zebrafish chordin mRNA to the ventral side of Xenopus embryos induced secondary axes. Ectopic overexpression in zebrafish resulted in an expansion of paraxial mesoderm and neurectoderm at the expense of more lateral and ventral derivatives, producing a range of defects similar to those of dorsalized zebrafish mutants (Mullins et al., 1996). In accordance with the proposed function of chordin in D-V patterning, dorsalized zebrafish mutants showed expanded domains of chordin expression by midgastrulation, while some ventralized mutants had reduced expression; however, in all mutants examined, early organizer expression was unaltered. In contrast to Xenopus, zebrafish chordin is also expressed in paraxial mesoderm and ectoderm and in localized regions of the developing brain, suggesting that there are additional roles for chordin in zebrafish embryonic development. Surprisingly, paraxial mesodermal expression of chordin appeared unaltered in spadetail mutants that later lack trunk muscle (Kimmel et al., 1989), while axial mesodermal expression was affected. This finding reveals an unexpected function for spadetail in midline mesoderm and in differential regulation of chordin expression during gastrulation.     

An outbreak of campylobacter food poisoning in a health care setting

An outbreak of campylobacter food poisoning in a group of health care workers is reported. The outbreak involved 12 of 31 staff members attending a departmental party. Investigations revealed that a chicken dish was the most likely vehicle of infection. The relative risk of developing symptoms after eating this dish was 6.15 (P < 0.002). Further investigation established that not only had the cooking instructions been misunderstood but storage of the the food the morning of the party did not comply with Department of Health guidelines. The outbreak was associated with considerable morbidity and cost. It is important that health care workers other than professional catering staff are aware of the guidelines available to ensure the correct preparation and storage of food if such events are to be held in hospital departments. It is also important that manufacturers of chicken products ensure that labelling regarding further cooking is clear and in large print.     

Experimental campylobacter infection and diarrhoea in immunodeficient mice

The responses of previously untested immunodeficient mouse strains to campylobacter infection are described. Three strains of adult immunodeficient mice (SCID-Beige, C.B-17-SCID-Beige and RAG-2) were inoculated intragastrically with Campylobacter jejuni NCTC 11168. All mice became heavily colonised, but none developed clinical signs of disease. Immunocompetent BALB/c mice inoculated similarly had much lower colonisation levels. The co-administration of iron dextran had no effect on colonisation levels nor the development of clinical signs of disease. In contrast, C.B-17-SCID-Beige mice, when inoculated with one of a series of 10 clinical isolates of C. jejuni, were more heavily colonised for extended periods (up to 5 months) and approximately 10-20% of the mice became ill with diarrhoea. C. jejuni was detected in mouse faeces throughout at levels of 10(7)-10(9) cfu/g. All mice killed whilst ill with diarrhoea displayed histopathological lesions typical of human campylobacteriosis. Severe pathology was limited to the large intestine and was suggestive of an acute, bacteria-induced inflammation. Although blood was detected in the diarrhoeal stools, no evidence of mucosal epithelial cell invasion was found by immunohistology. No pathology was detected in tissue sections from any of the animals that had not developed signs of disease following C. jejuni inoculation. These immunodeficient mouse strains are readily, and heavily, colonised as adults by C. jejuni. The diarrhoea, although sporadic, was reproducibly produced, and could provide the basis for pathogenicity studies.     

Nutrient regulation of gene expression: a methodological strategy

Nutrients should be viewed as the earliest of the hormone signals which allowed an organism to respond to changes in its nutrient environment. Defining nutrient function at the nuclear level will permit us to understand how our dietary environment is related to the development of nutritionally related pathophysiologies such as diabetes, heart disease, and cancer. Moreover, through the application of molecular techniques, we will begin to understand how specific nutrients govern the developmental pattern of specific organs such as the kidney. In this review, the fatty acid synthase gene is employed as a model to demonstrate how one sequentially approaches questions pertaining to the regulation of gene expression by a nutrient, and the article presents a nuclear explanation for how dietary polyunsaturated fats decrease blood triglycerides. More importantly, studies of this nature provide a basis for screening genetically susceptible populations and information that will allow the nutritionists of the 21st century to customize a diet for patients at risk.     

Outbreak of campylobacter enteritis in a residential school associated with bird pecked bottle tops

An outbreak of gastroenteritis caused by campylobacter infection was identified in May 1995 in a residential school in Gloucestershire for children with special needs. Eight primary and four secondary cases were identified, mostly confined to one house in the school; faecal specimens from three cases grew Campylobacter jejuni. A retrospective cohort study showed that drinking pasteurised milk from bottles with damaged tops was associated with illness in primary cases (p = 0.01). Bird pecking of milk bottle tops probably accounts for several thousand cases of human campylobacter infections during May and June each year in England and Wales. Milk bottle containers for doorstep delivery should be resistant to contamination by birds.     

Aminoacyl-tRNA synthetase gene regulation in Bacillus subtilis

In this review, we summarize progress on the regulation of the aminoacyl-tRNA synthetase genes in Bacillus subtilis. Most of the genes encoding this set of enzymes in B subtilis are members of a large family of Gram-positive genes and operons controlled by a novel antitermination mechanism that uses their cognate uncharged tRNA as the effector. A subset of these genes is, in addition, likely to be controlled at the level of mRNA processing and degradation. We describe the key experiments leading to these conclusions.     

Polyhedron-like inclusion body formation by a mutant nucleopolyhedrovirus expressing the granulin gene from a granulovirus

The polyhedrin gene in Bombyx mori nucleopolyhedrovirus (BmNPV) was replaced with the granulin gene of Trichoplusia ni granulovirus (TnGV). The substitution was verified by Southern hybridization, and expression of granulin by the mutant virus, BmGran, was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by amino acid sequencing of the predominant protein of BmGran inclusion bodies (IBs). Light and electron microscopy examination of BmGran-infected B. mori and BmN cells revealed large, cuboidal, polyhedron-like IBs in the nucleus and cytoplasm, but granules were not seen. IBs contained small, parallel, electron-dense streaks, which defined the geometric pattern of crystallization. Geometric patterns of nuclear IBs were frequently disrupted by occlusion of polyhedron envelope fragments, resulting in IB instability and fracturing. Virions were not embedded in most of the polyhedron-like IBs, but accumulated with polyhedron envelope fragments. Some virions were coated with matrix protein and were partially wrapped by polyhedron envelope. These results suggested that (1) the amino acid sequence of granulin insufficient for determining IB morphology in TnGV-infected cells, and TnGV may have genes, not present in BmNPV, that control granule formation, and (2) interactions among the virion, the IB envelope, and the matrix protein may be important in virion occlusion and IB morphology and stability.     

The transmission of campylobacter in piggeries; an epidemiological study

Epizootics of Rift Valley fever (RVF) are often associated with periods of heavy rainfall, which are favorable for mosquito vectors. However, in seasons with normal or low rainfall, enzootic circulation occurs, suggesting the existence of a natural host that can act as a cryptic carrier during interepizootic periods. To confirm the role of heavy rainfall in epizootic circulation, and to identify a possible natural host of RVF virus, serum samples from small terrestrial mammals in the Free State and Northern Cape regions of South Africa were collected before and after the 1988 floods. These areas are known to support epizootic circulation of RVF virus. The samples were tested for the presence of RVF virus-specific IgG using an ELISA and positive sera were confirmed by a neutralization test. Forty-seven (15%) of 312 Aethomys namaquensis (Namaqua rock rat) had antibodies to RVF virus. Of these positive sera, nine (6%) of 141 were collected before the floods of 1988 and 38 (22%) of 171 were collected afterwards (P = 0.001). Naive A. namaquensis were inoculated with RVF virus and developed a viremia, but no clinical symptoms, suggesting that they can act as temporary asymptomatic carriers of the virus. These results suggest a role for A. namaquensis as a cryptic carrier for RVF virus during interepizootic periods and support the results of other studies suggesting an amplifying role for heavy rainfall in the circulation of RVF virus.     

Plants transformed with a mutant alfalfa mosaic virus coat protein gene are resistant to the mutant but not to wild-type virus

Transgenic tobacco plants expressing the wild-type (wt) coat protein (CP) gene of alfalfa mosaic virus (AIMV) have been shown to be resistant to infection with viral particles and RNAs or to infection with viral particles only. The difference in resistance of these plants to RNA inocula was found to correlate with a difference in the expression level of the transgene. Plants expressing a mutant AIMV CP with the N-terminal serine residue changed to glycine have been shown to be susceptible to infection with wt viral particles or RNAs. By site-directed mutagenesis of AIMV cDNA a viable mutant virus encoding CP with the same N-terminal mutation was obtained. Plants expressing wt or mutant CP were resistant to the mutant virus, demonstrating that a single amino acid substitution in CP did not permit the virus to overcome CP-mediated resistance. Although the mutant CP did not confer resistance to wt virus when expressed in transgenic plants, it was still effective in classical cross-protection: plants infected with the mutant virus were resistant to severe strain of AIMV. A model to explain the data is discussed.