The Leeuwenhoek Lecture, 1997. Marek's disease herpesvirus: oncogenesis and prevention.

There are a number of neoplasias for which a herpesvirus is an essential part of the aetiology. Of these, Marek''s disease is the most common and provides excellent opportunities for the study of a herpesvirus-induced tumour both experimentally and under natural conditions in the field. Marek''s disease is caused by an alpha herpesvirus; it differs from the other oncogenic herpesviruses which are gamma herpesviruses. It is a ubiquitous virus in poultry populations of the world and is highly cell-associated and contagious, yet only a proportion of infected fowl develop tumours. Evidence is presented to suggest that at least one of the reasons for a wide variation in the incidence of the disease is a temporal interplay between virulent viruses and viruses of low or no virulence. The viral genes associated with the oncogenicity of Marek''s disease virus (MDV) are discussed and it is concluded that it is likely that several genes are involved. Finally, a brief history of vaccination to control Marek''s disease is given and mode of action discussed. It is concluded that the mechanism of protection is mainly through an antiviral cell mediated immune response, resulting in a lowered challenge virus burden. Marek''s disease viruses over the past 40 years have been evolving greater oncogenicity, some of which are not adequately controlled by the vaccines that are currently available. It is suggested that for MDV to produce tumours, there is a need for the cytolytic infection phase and that infection must be with an MDV which possesses a functional gC, ICP4 for maintaining latency which allows the expression of at least the 1.8 kb family, pp38, meq, and possibly pp14 genes, for maintaining the tumour state and possibly initiating this state. Intervention in this process reduces the chance of tumour formation and incidence in a population which can occur through natural or man-mediated infection with non-pathogenic MDVs.     

The Escherichia coli proteome: past, present, and future prospects.

Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects.     

The Leeuwenhoek Lecture, 1991. The influence of the host on microbes that cause disease.

Microbial pathogenicity or virulence, the capacity to cause disease, depends on microbial gene products that promote infection and penetration of mucous membranes, multiplication in the tissues, interference with host defence and sickness. Formation of these virulence determinants by microbes is influenced by the environment of the host, which differs from that in laboratory cultures. Studies of microorganisms grown in vivo, and of the host's influence on the production of virulence determinants, are increasing. In most studies, however, the complex conditions in vivo are not dissected to show the influence of particular factors. In future we should define specific host factors that are responsible for producing identified virulence determinants. There are three studies which point the way. Iron limitation in vivo causes production of bacterial siderophores, outer membrane receptors and some toxins. Erythritol, a growth stimulant for brucellae, causes intense placentitis and hence abortion in cattle, sheep and pigs. Cytidine 5'-monophospho-N-acetyl neuraminic acid (CMP-NANA) sialylates a conserved component of gonococcal lipopolysaccharide (LPS), thereby rendering gonococci in patients resistant to complement-mediated killing by serum. Although the lecture uses bacteria for examples, the principle applies equally to studies of viral and fungal pathogenicity.     

The use of biochemical markers, serotype and fimbriation in the detection of Escherichia coli clones

Biochemical reactions, O and K serotypes and presence of P-fimbriae were analysed in 116 Escherichia coli strains isolated in blood cultures from patients with bacteraemia and in 99 faecal strains isolated from healthy individuals. By using biochemical typing, the strains could be grouped into six main clusters with similarity index less than 0.8 (Gower, 1971) and altogether 16 subclusters with similarity index 0.82-0.89. The most discriminating tests between the clusters were fermentation of D-tagatose, saccharose, salicin and sorbose. No single biochemical property could differentiate bacteraemic isolates from faecal strains, although strains isolated from blood were significantly more often found in certain subclusters, whereas other subclusters contained mainly control strains. Bacteraemic strains possessed P-fimbriae more often, especially strains isolated from patients with E. coli in the urine concomitantly with bacteraemia. Equally, no single reaction could separate P-fimbriated from non-P-fimbriated strains. D-Tagatose was fermented more often by the P-fimbriated strains; on the other hand, melibiose and lactose fermentation tests were less often positive. Certain O serotypes (O1, O4, O6, O7, O18 and O25) were more common among bacteraemic isolates than controls. K serotypes such as K1, K5 and K52 were also more frequent among blood isolates. We conclude that a combination of biochemical tests, fimbriation and serotyping might be used to identify potentially pathogenic clusters of E. coli.     

The Leeuwenhoek Lecture 2000 the natural and unnatural history of methane-oxidizing bacteria

Methane gas is produced from many natural and anthropogenic sources. As such, methane gas plays a significant role in the Earth's climate, being 25 times more effective as a greenhouse gas than carbon dioxide. As with nearly all other naturally produced organic molecules on Earth, there are also micro-organisms capable of using methane as their sole source of carbon and energy. The microbes responsible (methanotrophs) are ubiquitous and, for the most part, aerobic. Although anaerobic methanotrophs are believed to exist, so far, none have been isolated in pure culture. Methanotrophs have been known to exist for over 100 years; however, it is only in the last 30 years that we have begun to understand their physiology and biochemistry. Their unique ability to use methane for growth is attributed to the presence of a multicomponent enzyme system-methane monooxygenase (MMO)-which has two distinct forms: soluble (sMMO) and membrane-associated (pMMO); however, both convert methane into the readily assimilable product, methanol. Our understanding of how bacteria are capable of effecting one of the most difficult reactions in chemistry-namely, the controlled oxidation of methane to methanol-has been made possible by the isolation, in pure form, of the enzyme components.The mechanism by which methane is activated by sMMO involves abstraction of a hydrogen atom from methane by a high-valence iron species (FeIV or possibly FeV) in the hydroxylase component of the MMO complex to form a methyl radical. The radical combines with a captive oxygen atom from dioxygen to form the reaction product, methanol, which is further metabolized by the cell to produce multicarbon intermediates. Regulation of the sMMO system relies on the remarkable properties of an effector protein, protein B. This protein is capable of facilitating component interactions in the presence of substrate, modifying the redox potential of the diiron species at the active site. These interactions permit access of substrates to the hydroxylase, coupling electron transfer by the reductase with substrate oxidation and affecting the rate and regioselectivity of the overall reaction. The membrane-associated form is less well researched than the soluble enzyme, but is known to contain copper at the active site and probably iron.From an applied perspective, methanotrophs have enjoyed variable successes. Whole cells have been used as a source of single-cell protein (SCP) since the 1970s, and although most plants have been mothballed, there is still one currently in production. Our earlier observations that sMMO was capable of inserting an oxygen atom from dioxygen into a wide variety of hydrocarbon (and some non-hydrocarbon) substrates has been exploited to either produce value added products (e.g. epoxypropane from propene), or in the bioremediation of pollutants such as chlorinated hydrocarbons. Because we have shown that it is now possible to drive the reaction using electricity instead of expensive chemicals, there is promise that the system could be exploited as a sensor for any of the substrates of the enzyme.     

The biochemical and genetic basis for high frequency thiomethyl galactoside resistance in lambda,lambdadg lysogens of Escherichia coli.

In a culture of Escherichia coli K12 gal (lambdadg), cells which form large colonies on agar plates containing galactose and thiomethyl beta-D-galactoside (TMG) appear at high frequency. These clones are resistant to growth inhibition by TMG on galactose minimal medium. Biochemical studies of the steady-state levels of galactokinase and UDPgalactose 4-epimerase suggest that the resistant clones have extra copies of the genes for the galactose-metabolizing enzymes. The mutation for TMG resistance is not located in either the bacterial or the bacteriophage genome, but is probably due to an aberrant association between cell and prophage DNA. Mapping the TMG-resistant characteristic by phage P1 indicates that TMG-resistant bacteria posses at least two GAL+ OPERONS, ONE OF WHICH IS COTRANSDUCIBLe with bio+. In addition, TMG-resistant bacteria behave like lambdadg polylysogens when challenged with the phage lambdaI90c17. From these genetic experiments we conclude that TMG-resistant bacteria arise by duplication of the lambdadg prophage. Finally, gal+ bacteria which carry a single, additional, lambdadg prophage are TMG-resistant. TMG resistance is probably a gal+ gene dosage effect.     

The art and design of genetic screens: Escherichia coli

This article summarizes the general principles of selections and screens in Escherichia coli. The focus is on the lac operon, owing to its inherent simplicity and versatility. Examples of different strategies for mutagenesis and mutant discovery are described. In particular, the usefulness and effectiveness of simple colour-based screens are illustrated. The power of lac genetics can be applied to almost any bacterial system with gene fusions that hook any gene of interest to lacZ, which is the structural gene that encodes beta-galactosidase. The diversity of biological processes that can be studied with lac genetics is remarkable and includes DNA metabolism, gene regulation and signal transduction, protein localization and folding, and even electron transport.     

The Florey Lecture, 1991. The colony-stimulating factors: discovery to clinical use.

The four colony-stimulating factors, GM-GSF, G-CSF, M-CSF and Multi-CSF, are specific glycoproteins with a likely common ancestral origin which interact to regulate the production, maturation and function of granulocytes and monocyte-macrophages. Each has been purified and produced in active recombinant form. Animal studies have shown the ability of injected CSF to increase the production and functional activity of granulocytes and macrophages in vivo and to enhance resistance to infections. These studies have led to the current extensive clinical use of CSFs to promote the formation and function of granulocytes and macrophages in a wide variety of disease situations in which there is an associated risk of serious infections. Although our knowledge of the control of haemopoiesis remains incomplete, the approaches used to develop the CSFs can be used to extend this knowledge, with the promise of the introduction into clinical medicine of additional effective therapeutic agents.     

Millets genetic engineering: the progress made and prospects for the future

Millets comprise a highly variable small-seeded group of Poaceae members that can grow in extreme environmental conditions of drought, high temperature and low soil fertility hence, recognized as climate-resilient. Among millets, the phylogenetic closeness of Setaria with other agronomically important grasses like maize, sugarcane, and sorghum helped in its adoption as a translational model plant. Established efficient gene transfer methodology is a prerequisite for embracing plant species as models. However, genetic engineering of some of the economically important millets has been started in the 1990s, but inadequate progress made this group lag behind other members of Poaceae as rice, maize and wheat. Genetic transformation in millets has generally been achieved by a physical method of microprojectile bombardment, recently Agrobacterium-mediated gene transfer technique has also established in some of the millets but with very few reports. The central hindrance in millet transformation is its recalcitrant nature to regeneration through tissue culture techniques. Optimization of highly efficient regeneration procedure for each millet species is thus, necessary to establish advanced transformation system for them. The possibility of alternative transformation approaches is also discussed. The establishment of robust gene transfer methods whether it’s conventional in-vitro tissue culture dependent or in-planta are important for functional validation studies and would enable development of crop improvement strategies. This review presents the progress made on millet genetic transformation, discussing the major challenges that need to be overcome and future opportunities of transgenic techniques in various millets.

     

Genetic and biochemical requirements for chemotaxis to L-proline in Escherichia coli.

Chemotaxis to L-proline was examined by the capillary assay, using a set of Escherichia coli strains bearing well-defined defects in the enzymes of proline transport and utilization. Aspartate taxis was measured as a constitutive, control activity whose receptor and transducer requirements are known. Proline chemotaxis showed a pattern of induction more analogous to that of proline dehydrogenase than of that of proline transport, but chemotaxis to proline was eliminated by mutations eliminating either or both of these activities. No response to proline was observed in the absence of a proline concentration gradient or when succinate was provided as an oxidizable carbon source. These data suggest that the chemotactic response to proline results from a direct impact of proline oxidation on the energy metabolism of the cell.     

Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli.

The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310, 1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemical analysis of the K5 capsular polysaccharide. Whereas the K5 clone expressed the K5 polysaccharide as a well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparations and ultrathin sections of the expression mutants. Immunogold labeling of sections from the region 3 mutant revealed the capsular K5 polysaccharide in the cytoplasm. With the region 1 mutant, the capsular polysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide, the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria. Polysaccharides were isolated from the homogenized mutants with cetyltrimethylammonium bromide. The polysaccharide from the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and both polysaccharides were substituted with phosphatidic acid. The polysaccharide from the region 3 mutant was smaller and was not substituted with phosphatidic acid. These results prompt us to postulate that gene region 3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membrane and that region 1 directs the transport of the lipid-substituted capsular polysaccharide through the periplasm and across the outer membrane.     

Dihydroneopterin triphosphate epimerase of Escherichia coli: purification, genetic cloning, and expression.

The enzyme catalyzing the epimerization at position 2' of dihydroneopterin triphosphate was purified by a factor of about 10,000 from cell extract of Escherichia coli. The cognate gene was cloned, sequenced, expressed, and mapped to kb 2427 on the E. coli chromosome.     

The bulged nucleotide in the Escherichia coli minimal selenocysteine insertion sequence participates in interaction with SelB: a genetic approach

The UGA codon, which usually acts as a stop codon, can also direct the incorporation into a protein of the amino acid selenocysteine. This UGA decoding process requires a cis-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stem-loop structure. In Escherichia coli, selenocysteine incorporation requires only the 17-nucleotide-long upper stem-loop structure of the fdhF SECIS. This structure carries a bulged nucleotide U at position 17. Here we asked whether the single bulged nucleotide located in the upper stem-loop structure of the E. coli fdhF SECIS is involved in the in vivo interaction with SelB. We used a genetic approach, generating and characterizing selB mutations that suppress mutations of the bulged nucleotide in the SECIS. All the selB suppressor mutations isolated were clustered in a region corresponding to 28 amino acids in the SelB C-terminal subdomain 4b. These selB suppressor mutations were also found to suppress mutations in either the loop or the upper stem of the E. coli SECIS. Thus, the E. coli SECIS upper stem-loop structure can be considered a "single suppressible unit," suggesting that there is some flexibility to the nature of the interaction between this element and SelB.     

Escherichia coli F41 adhesin: genetic organization, nucleotide sequence, and homology with the K88 determinant.

The genetic organization of the polypeptides required for the biosynthesis of the F41 adhesin of enterotoxigenic Escherichia coli strains was investigated. Maxicell analysis demonstrated that a recombinant plasmid which mediated mannose-resistant hemagglutination and F41 antigen production encoded four polypeptides of 29, 30, 32, and 86 kilodaltons. The 29-kilodalton protein was identified as the F41 antigen, and the nucleotide sequence of the gene was determined. Extensive homology was observed between the region encoding the putative signal sequences of the F41 and K88 antigens and in the region immediately upstream of the antigen genes. The nucleotide sequence homology between F41 and K88 determinants was further investigated by Southern blot hybridization. A K88 probe hybridized at high stringency to all fragments shown to be essential for F41 production except for fragments internal to the F41 antigen gene.