One of the things I have always been curious about is why vaccines are effective for certain viruses and not others. The major viral scourge of today is, of course, HIV, with influenza probably a close second. Yellow fever was a major problem in the world until the early 1930s. In the case of HIV, a vaccine has not yet been created, despite an investment of hundreds of millions of dollars. An effective yellow fever vaccine was developed some 75 years ago, in the early 1930s. In fact, since then, nearly 300 000 000 doses of the latter vaccine have been administered without adverse effect. The question I am going to ask in this essay is, why has there been a vaccine success for one of these viruses, but not the other? In a curious way, the answer is decidedly ecological.

The primary sources for my information came from several very good virology books, discussions with a virologist colleague, plus some literature searches in our library. The first tome I used was Topley and Wilson's Volume I of Microbiology and Microbial Infections: Virology, edited by Brian Mahy and Leslie Collier (1998). A second was a popular general ecology textbook, Evolutionary Analysis, written by Scott Freeman and Jon Herron (2004). I also had a series of very productive discussions with Pat Lord, a very solid virologist in our Biology Department here at Wake Forest University.

Since I have never had malaria, all I know about rigor, or the high fever that follows, is what I read from those who have had the experience. Not long ago, in preparing to write this essay, I spent some time interviewing Robert Desowitz, the American parasitologist who has so marvelously written about his many experiences working with tropical diseases over the years, e.g., The Malaria Capers, Who Gave Pinta to the Santa Maria, etc. I asked Bob if, while living so long in so many exotic parts of the world, he had ever come down with malaria, or any of the other tropical diseases about which he had written so much or done so much research. He said that he had not been infected with anything except Plasmodium vivax and Plasmodium falciparum, that both of them came at the same time, and by accident! Acquiring malaria by accident is ironic in his case because he had spent nine years at the West African Trypanosome Research Lab in Jos, in the northern part of Nigeria, working on trypanosomes in the employ of the British Colonial Service. He then spent five more years as Chairman of the Department of Parasitology at the University of Singapore, plus several more years in southeastern Asia while with the U.S. Army at their SEATO (Southeast Asia Treaty Organization) lab in Bangkok, Thailand.

Recognition of the biological nature of sulphate reduction in natural environments, and identification of the bacterial species involved dates to the latter part of the nineteenth century, and the seminal work of such giants of the early days of microbiology as Beijerinck and Winogradsky. The central role of environmental studies in highlighting the issues to be addressed and the problems to be solved, has remained to this day a constant theme in microbiological analyses of the sulphate reducers.

The modern era of such analyses, however, can be said to date from the period around 1960 when the demonstrations by Postgate and Peck, respectively, of the presence of cytochromes and of phosphorylation linked to anaerobic respiration in sulphate-reducing bacteria (SRB), fundamentally altered our view of the biochemical nature of these organisms and, in particular, of their mechanisms of energy conservation.

There then followed a period of intense activity centred on: elucidation of the metabolic pathways of substrate utilisation and the mechanisms of energy generation; cultural techniques and the identification of an ever-increasing number of new species; and the appreciation of their significant role in maintaining, or disrupting, the biological balance of many natural and man-made ecosystems.

These themes of biochemistry and cell physiology, phylogeny, and ecology remain central to the understanding of SRB themselves, and of their interactions with other components of the biosphere. In recent years, however, their study has undergone a further paradigm shift with the introduction of the many powerful experimental techniques and analytical approaches of molecular biology.

Any book of the present sort requires a large number of sources, and I sought as many as I could. First, there were those folks who took the time to sit and talk with me. Without exception, they all opened up and answered every question I asked. But far more than this, they helped lead me into areas that I did not know about or might not have otherwise probed. Second, I want to thank them for all of the reprints they generously provided so that I could ‘bone up’ on their areas of interest before the interview was consummated. Third, there are several books that describe the fascinating history of parasitology. I am confident I have given appropriate credit for all that I used as I went along. There are, however, several special authors and books that I want to emphasize and from which I drew invaluable information. These of course include Bob Desowitz and all of his popular tomes, but especially those that dealt with the history of malaria and its treatment, primarily The Malaria Capers. Mark Honigsbaum's The Fever Trail was an extraordinary account of the history of quinine and the cinchona tree from which this herbal remedy comes. It should be read by anyone with an interest in malaria. I also read a large number of general historical accounts dealing with our discipline. The best was A History of Human Helminthology, by D. I. Grove.

Whenever my wife and I go to London, we always stay at the same hotel in Kensington. Our favorite pub, The Goat, is nearby on Kensington High Street. Not too long ago, I recall sitting with Pete Olson and one of his postdocs in our pub enjoying a pint of lager. Pete, his postdoc, and I were talking about the Journal of Parasitology and some of what goes on in terms of editing. During the conversation, I mentioned that the only really contentious issues that have arisen over the years generally involved confrontations between referees and authors concerned with systematics/phylogenetics papers. I said to Pete, “Aside from natural personality clashes between certain folks, why do you suppose this is the case?” Pete responded immediately, like he knew the question was coming and had prepared an answer in advance. He said, “This is the only area of parasitology where opinion is acceptable in print form.”

When Pete said this, I knew exactly what he meant, because when one thinks about it, a genus and species is really nothing more than a hypothetical construct. It is an opinion, well considered and documented in most cases, but still an opinion. When dealing with hypotheses, people will often have different ideas about them.

INTRODUCTION

Toxic heavy metals and metalloids constitute an international pollution problem that not only impacts public health but also is of environmental and economic importance. Prokaryotes with the physiological activity of sulphate reduction are found in a number of environmental sites containing toxic metals and these microorganisms have developed several different strategies for resistance to toxic elements. Some bacteria have developed detoxification strategies that are potentially useful for bioremediation. Since sulphate-reducing bacteria (SRB) are found in a large number of contaminated sites containing toxic metals, it is apparent that these organisms have a functional defence system that enables them to persist and even grow under metal stress. The enzymatic metal reduction by SRB offers an alternative to chemical processes to remediate environments containing redox-active toxic metals and metalloids. While Hockin and Gadd discuss in Chapter 14 the bioremediation activities of sulphate-reducing bacteria, this chapter focuses on the enzymatic processes associated with metal reduction. We review results obtained with isolated proteins and discuss the potential of sulphate-reducers by reviewing putative proteins found in their genomes. Reference is made to putative genes present in Desulfovibrio (D.) vulgaris strain Hildenborough (Heidelberg et al., 2004), D. desulfuricans strain G20 (http://www.jgi.doe.gov), Desulfotalea (Des.) psychrophila (Rabus et al., 2004) and Archaeoglobus (A.) fulgidus (Klenk et al., 1997).

ENZYMATIC ACTIVITIES INVOLVING REDOX-ACTIVE ELEMENTS

The detoxification of an environment arising from SRB reductions is considered by many as an important event for bioremediation of various polluted environments In addition to precipitation of metals by biogenic hydrogen sulfide, the SRB are highly capable of reducing many soluble redox-active elements.

INTRODUCTION

Besides their challenging and ancient energy metabolism, and applied relevance, much of the interest in sulphate-reducing bacteria arises from their ecophysiological significance in marine environments (Widdel, 1998). In the biologically highly active shelf sediments they contribute to more than 50% of organic carbon remineralization (Jørgensen, 1982), which can only be explained by complete substrate oxidation (Fenchel and Jørgensen, 1977). While this capacity is not present among the frequently isolated and intensively studied Desulfovibrio spp., it could be demonstrated with e.g. the newly isolated Desulfobacter postgatei (Widdel and Pfennig, 1981) and Desulfobacterium autotrophicum (Brysch et al., 1987). The latter employs the C1/CO-dehydrogenase pathway for complete oxidation of acetate to CO2 as well as for CO2-fixation (Schauder et al., 1989). Most of the known sulphate-reducing bacteria can be grouped into the two deltaproteobacterial families Desulfovibrionaceae (Devereux et al., 1990) or Desulfobacteriaceae (Widdel and Bak, 1992). This phylogenetic distinction is to a large extent paralleled by the capacities for incomplete (to acetate) and complete (to CO2) oxidation of organic substrates, respectively.

At present, more than 450 prokaryotic genomes have been completely sequenced and about 1000 further prokaryotic genomes are in progress (http://www.genomesonline.org). While most genome projects primarily reflect biotechnological or biomedical research interests, environmentally relevant microorganisms have been selected for genome sequencing projects only during the last few years. This chapter provides an overview of the technologies involved and of the current status of genomic research with sulphate-reducing prokaryotes.

PRESENCE OF SULPHATE-REDUCING BACTERIA IN OXIDISED HABITATS

During the second half of the nineteenth century the formation of sulphide from sulphate was recognised as a biogenic process (Meyer, 1864). While it was initially suggested that algae were the catalysing organisms (Cohn, 1867), Hoppe-Seyler demonstrated in 1886 that the process required anoxic conditions and was chemotrophic, requiring external electron donors. In 1895, Beijerinck proved that sulphate reduction is catalysed by bacteria and described the first pure culture, Spirillum desulfuricans. This organism was described as strictly anaerobic and was irreversibly inhibited by oxygen.

The view that sulphate-reducing bacteria (SRB) are extremely sensitive to oxygen started to change in the late 1970s when sulphate reduction was demonstrated to occur also in oxidised sediment layers which showed no traces of FeS and were considered oxic (Jørgensen, 1977). Similarly, cultivation-based studies revealed the presence of viable sulphate reducers within these layers (Laanbroek and Pfennig, 1981; Battersby et al., 1985; Jørgensen and Bak, 1991). However, it was found that oxygen did not penetrate as deep into sediments as previously assumed and that large parts of the oxidised, hence FeS-free layers, were in fact anoxic. In sediment layers that contain oxidised manganese or iron species, sulphide can be chemically reoxidised to elemental sulphur (Aller and Rude, 1988), or, in the case of manganese oxide, even to thiosulphate (Schippers and Jørgensen, 2001).

INTRODUCTION

Sulphate-reducing bacteria (SRB) are anaerobes, which derive energy for growth from anaerobic metabolism, coupling the oxidation of organic substrates with the dissimilatory reduction of sulphate to hydrogen sulphide (sulphate respiration). Although generally considered as strict anaerobes, more and more data indicate a higher abundance and metabolic activity in oxic zones of biotopes, such as marine and freshwater sediments, than in neighbouring anoxic zones (Ravenschlag et al., 2000; Sass et al., 1997; 1998). A well-documented example of sulphate reduction under oxic conditions is also provided by cyanobacterial mats. Here a zone of photosynthetic oxygen synthesis overlaps with a zone of sulphide production by SRB and a zone of oxygen-dependent microbial sulphide oxidation, creating steep, opposing gradients of oxygen and sulphide, which fluctuate with the rhythm of day and night (Canfield and des Marais, 1991; Teske et al., 1998; Caumette et al., 1994). In cyanobacterial mats from the saline evaporation pond in Baja California, Desulfobacter and Desulfobacterium are restricted to greater depths while the Desulfococcus and Desulfovibrio groups are predominant in the upper part of the photo-oxic zone (Risatti et al., 1994). The high numbers of SRB found in these oxic environments indicate that these organisms are able to deal with temporal exposures to oxygen concentrations as high as 1.5 mM (Sigalevich and Cohen, 2000).

In these oxygen-exposed systems SRB of the genus Desulfovibrio are among the most oxygen-tolerant, e.g. Desulfovibrio oxyclinae was isolated from Solar Lake microbial mats (Krekeler et al., 1997) and Desulfovibrio desulfuricans strain DvO1 was isolated from activated sludge aerated to atmospheric oxygen saturation (Kjeldsen et al., 2005).

Several weeds such as the bindweeds Convolvulus arvensis and Calystegia sepium, which belong to the economically most important weeds, are difficult to control with conventional physical and chemical methods. Biological control with fungal biocontrol agents (FBCA) offers a promising alternative. However, the employment of such FBCAs implies not only the identification of highly effective strains and the development of cheap formulation and production techniques, but also an extensive risk assessment including tracking of FBCAs and their toxic metabolites in the environment. Stagonospora convolvuli strain LA39 effectively controls field and hedge bindweed in the field. For this strain formulation and application techniques were developed and currently markers for environmental tracking of the fungus are being designed. A major metabolite produced by LA39 is elsinochrome A (ELA). To assess the risk of ELA production by LA39, different methods, such as toxicity tests, stability tests and methods for environmental monitoring, were developed. Our results revealed that ELA is toxic to a wide array of organisms. However, ELA was never detected in LA39 treated bindweed or crop plants, and the ELA content in the applied biocontrol product is far too low to result in a toxic concentration in the environment. Furthermore, ELA is quickly degraded by sunlight. In conclusion, LA39 is a promising candidate for the development of a successful biocontrol product against bindweed. Harmful side effects of ELA production can be excluded.

Introduction

To apply suitable bioremediation techniques, an understanding of the physical, chemical and biological attributes of an environmental matrix is required. Effective bioremediation is based on optimizing these attributes to enhance the biodegradation of target pollutants. Fundamental to these processes is the concept of bioavailability and bioaccessibility of these pollutants at a suitable and relevant scale (Alexander, 2000; Semple et al., 2004). Environmental analyses are still based on chemical approaches that usually require an exhaustive extraction step prior to chromatographic analysis. This extracted fraction is commonly modelled to assess the portion that may cause harm to a particular target receptor. It is widely acknowledged that modelled values may be appropriate for human risk assessment (though inherently conservative) but yield little information for hazard assessment in a wider ecological or environmental context (Alexander, 2000). Many authors have demonstrated that chemical analysis alone does not provide information regarding the bioavailable fraction of compounds nor about their effects on selected biological receptors (Power et al., 1998; Hansen & Sørensen, 2001; Belkin, 2003; Paton & Killham, 2003). Biological assays are able to complement chemical analysis by considering the effects of all pollutants present in a sample, including those not detected by chemical analysis or those unable to be fitted in a model. Bioassays are used for monitoring the progress of bioremediation because they determine the bioavailable fraction of compounds that in part determines the biodegradability of a compound (Hansen & Sørensen, 2001; Paton & Killham, 2003). However, Semple et al.

Introduction

In the periodic table, about 75% of the elements are classified as metals. While many metals are essential, e.g. K, Na, Ca, Mn, Mg, Fe, Cu, Zn, Co and Ni, many others have no apparent essential functions, e.g. Rb, Cs, Al, Cd, Ag, Au, Sr, U, Hg, Pb (Gadd, 1993). However, all can interact with fungi and most of them can exhibit toxicity above a certain threshold concentration, which will vary depending on the organism, metal species and environmental factors (Gadd & Griffiths, 1978; Gadd, 1993).

The redistribution of toxic metals in the biosphere as a result of human activity has become an important process in the biogeochemical cycling of these metals. The main source of metal pollution is industrialization including operational and former mining sites, foundries and smelters, untreated sewage sludge, and as diffuse sources, metal piping, traffic, combustion by-products from coal-burning power stations, and other atmospheric pollution. Arsenic, cadmium, chromium, copper, nickel, lead and mercury are the most common metal pollutants (Knox et al., 2000) with a selection of metal radionuclides also entering the environment from a number of sources (Gadd, 2005).

Unlike degradable organic contaminants, metals are not decomposed in the environment. They can, however, be buried (or otherwise contained), removed and recycled, or transformed into less toxic forms (Knox et al., 2000), these processes employing physical, chemical and biological methods singly or in combination.

Abstract

Trichoderma-based biofungicides are a reality in agriculture, with more than 50 formulations available today as registered products worldwide. Several strategies have been applied to identify the main genes and compounds involved in this complex cross-talk between the fungal antagonist and the microbial pathogen, as mediated by the plant. Proteome and genome analysis have greatly enhanced our ability to conduct holistic and genome-based functional studies. We have identified and determined the role of a variety of novel genes and gene-products, including ABC transporters, enzymes and other proteins that produce or act as novel elicitors of induced systemic resistance, proteins recognized by the plant as avirulence factors, as well as molecules that generally activate the antagonistic activity in Trichoderma spp. We have cloned mycoparasitism-related promoters and used them in combination with GFP and other markers to study the interaction in vivo and in situ between Trichoderma and the fungal pathogen or the plant. Finally, we have transgenically improved the ability of the antagonist to kill other microbes and to activate plant defence mechanisms.

Introduction

Plant diseases caused by pathogenic fungi infections represent a major limiting factor for the cultivation and the conservation of agricultural plants of interest. The consequences of parasite attack result in both quantitative and qualitative reduction of crop production, large economic losses and represent a risk for human and animal health due to the accumulation of residues in the environment and mycotoxin contaminants in food products.

Introduction

Overview

This paper explores the mechanisms of metal toxicity towards cells, specifically some advances being made in this area through work with the unicellular fungus, Saccharomyces cerevisiae. The two principal questions addressed here are:

1. Is oxidative damage the cause of cellular metal toxicity?

2. Why do individual cells exhibit widely differing metal resistances?

Several powerful experimental tools are unique to S. cerevisiae among eukaryotes, and are being exploited to help elucidate the mechanism(s) of metal toxicity. Furthermore, in conjunction with its unicellular morphology, S. cerevisiae provides an ideal system with which to explore the topical problem of cell individuality, applied here to metal toxicity. This chapter provides an overview of these fields, illustrated with key findings from the author's laboratory.

Metals in the environment and relevance to fungi

A wide range of industrial activities give rise to metal pollutants, which continue to be released into the environment at potentially harmful levels. Localized concentration of certain metals may also arise naturally. For example, toxic levels of the biologically essential metal copper are often associated with certain mineral ores as well as industrial or agricultural discharges. Cadmium is used widely in electroplating and galvanizing industries, as a colour pigment in paints and in batteries, and as a by-product of zinc and lead mining and smelting. Zinc, lead and other metals also may be released from similar types of sources.

Introduction

Epichloë endophytes (Epichloë/Neotyphodium species) are an important group of clavicipitaceous fungi that form symbiotic associations (symbiota) with temperate grasses of the Pooideae subfamily (Scott, 2001a; Schardl, Leuchtmann & Spiering, 2004). These biotrophic fungi systemically colonize the intercellular spaces of leaf primordia, leaf sheaths and culms of vegetative tissue and the inflorescence of reproductive tissues. The asexual Neotyphodium species have no external growth stage and consequently form symptomless associations with their grass host where they are vertically transmitted through the seed following colonization of the developing ovule. Most of the sexual Epichloë species are also transmitted vertically but because of their ability to form external stromata on inflorescence tissue, can also be transmitted horizontally (Leuchtmann & Schardl, 1998; Schardl & Leuchtmann, 1999). The mating system is heterothallic (outcrossing) and in nature is mediated by anthomyiid flies (Botanophila spp.), which transfer spermatia between stromata (Bultman et al., 1995; Bultman et al., 1998). Formation of perithecia and release of ascospores into the environment provides a source of vegetative hyphae that give rise to conidia that subsequently infect new hosts, probably by way of colonization of the stigma and style of the host inflorescence (Chung & Schardl, 1997).

Taxonomy of epichloë endophytes

At least nine different Epichloë species are recognized including E. typhina, a broad host range species (Schardl & Wilkinson, 2000) and E. festucae, a natural symbiont of Festuca spp. (Leuchtmann, Schardl & Siegel, 1994). E. festucae is also capable of forming compatible associations with perennial ryegrass, Lolium perenne (Christensen et al.

Introduction

Rice blast disease, caused by the filamentous fungus Magnaporthe grisea, is a serious and recurrent problem in all rice-growing regions of the world (Talbot, 2003; Valent & Chumley, 1991). It is estimated that each year enough rice is destroyed by rice blast disease to feed 60 million people. Control of this disease is difficult; new host-specific forms develop quickly to overcome host resistance and chemical control is typically not cost effective (Ou, 1987). Infections occur when fungal spores land and attach themselves to leaves using a special adhesive released from the tip of each spore (Hamer et al., 1988). The germinating spore develops an appressorium, a specialized infection cell, which generates enormous turgor pressure – up to 8 MPa – that ruptures the leaf cuticle allowing invasion of the underlying leaf tissue (de Jong et al., 1997; Dean, 1997). Subsequent colonization of the leaf produces disease lesions from which the fungus sporulates and spreads to new plants. When rice blast infects young rice seedlings, whole plants often die, while spread of the disease to the stems, nodes or panicle of older plants results in nearly total loss of the rice grain. Recent reports have further shown that the fungus has the capacity to infect plant roots (Sesma & Osbourn, 2004). Different host-limited forms of Magnaporthe also infect a broad range of grass species including wheat, barley and millet.