Preface

Environmental microbiology is currently one of the most rapidly expanding areas of scientific research. Impetus for advanced investigations has been provided by the development and application of molecular techniques that facilitate the identification, characterization and monitoring of microbes. These advances now allow detailed investigations, developed in the laboratory, to be undertaken in the natural environment. Such studies confirm the remarkable biological diversity represented by microorganisms from their basic genetic structure to the regulated communications that occur within and between communities contributing to ecosystem function. However, while it is apparent that microorganisms constitute the greater part of the planet's biomass, and are central in maintaining the biosphere, we remain essentially ignorant of a great majority of the functions or processes they undertake. One of the limiting factors in the study of their ecology, even within communities or populations, is that of scale. For instance, for soil it is very hard to assess microbes and their activities at the level of each individual pore where microbial soil inhabitants occur. Highly sensitive and specific tools are, thus, required for such detailed investigations. However, there is a paradox. Until a greater knowledge of the genetic and metabolic diversity within the microbial environment is obtained it remains difficult to investigate these complex communities or design relevant experiments that target the role of individuals or specific genes and their products. This situation is currently changing at an ever increasing rate. In this volume we have attempted to bring together a series of reviews of the approaches taken to study the ecology and functional activity of individual microbial cells and populations in environmental habitats, with a special focus on the use of marker/reporter genes for monitoring release strains.

Traditionally, microbial ecology has been limited to studies of microbial processes, such as cycling of nitrogen or carbon, occurring by uncharacterized species in a "black box" scenario. For example, denitrifi-cation, nitrification and nitrogen fixation processes have been assessed by analyzing the specific nitrogen forms appearing as a result of these processes. On the other hand, the study of particular taxa was limited to those for which suitable cultivation methods and laboratory growth media had been devised. However, we now know that the majority of microorganisms in nature are not capable of growing on the available media under standard laboratory conditions. Moreover, in some instances bacteria, that have been successfully cultivated in the laboratory, may lose the ability to grow on laboratory media after introduction into the environment, presumably as a result of a stress response. These bacteria may still be viable or metabolically active in the environment and this apparently recalcitrant state must be considered when these organisms are studied. The ability to persist and form a "resting stage" may be ecologically significant in niche exploitation. The phenomenon of viable but non-culturable (VBNC), as described for Vibrio spp, is discussed in Chapter 1.

In the 1980s the need to identify and track specific microorganisms in environmental samples was highlighted by the requirement for risk assessment of genetically modified microorganisms (GMMs) that were to be deliberately released into natural settings. The current status of regulatory requirements for GMMs is discussed in Chapter 12. For risk assessment of GMMs it is no longer possible to rely purely on traditional monitoring methods since the specific GMM has to be distinguished from its parent, or wild type, strain that could also inhabit the same ecosystem. Based on the requirement for specific monitoring methods, a variety of molecular tools have been developed to unambiguously track GMMs in the environment. These tools and examples of their applications for tracking GMMs in field studies are described in this book. The molecular tools range from nucleic acid-based monitoring methods (Chapter 3) to the use of marker genes (biomarkers) with distinct phenotypes that serve as tags for identification of specific microbes in the laboratory (Chapters 5-7) and in the natural environment (Chapters 8-11).

Following the established method of isolating defined strains using selective bacteriological media, nucleic acid-based molecular tools were the first to be applied for the purpose of directly tracking GMMs in environmental samples. This methodology was dependent on the ability to isolate nucleic acids from the environment, and a series of methods were developed for the extraction of DNA and more recently RNA, from complex matrices such as soil. These methods have been continually refined since the first published examples (reviewed in Chapter 3). As the technology improves, further optimizations in sensitivity and specificity can be expected. A variety of nucleic acid extraction methods were originally optimized for the different environmental sample types under analysis. These extraction and analytical methods have now been simplified and applied as essentially standard protocols for tracking GMMs in any environment. In particular, the application of polymerase chain reaction amplification (PCR) has revolutionized nucleic acid-based tracking methods by substantially increasing the sensitivity of detection over nucleic acid hybridization methods (Chapter 3).

A biomarker can be defined as a DNA sequence, introduced into an organism, which confers a distinct genotype or phenotype to enable monitoring in a given environment. In some cases, an intrinsic marker is sufficient for monitoring a particular bacterial species. An intrinsic marker is a nonintroduced DNA sequence or a natural genotype that serves as a signature for a particular organism or group of organisms. Intrinsic markers are further described in Chapters 2 and 4. Chapter 2 discusses antibiotic resistance as a special type of intrinsic marker. Usually, intrinsic markers are not sufficiently specific for tracking of GMMs, since they are also present in the parent, or "wild-type", strain, or they may be prevalent in the environment under study. For some applications, a reporter gene (bioreporter) can be used. A bioreporter is a gene encoding an easily detectable phenotype that can be used to measure gene expression when fused to appropriate promoter target sequences. The choice of biomarker or bioreporter depends on the particular strain, the environment studied and the questions to be addressed. Several of the most promising monitoring methods and examples of their application are described in detail in Chapters 5-7 of this book. In Chapters 8-11, examples are given of the use of these biomarkers to track specific GMMs after release during field trial experiments. Chapter 12 outlines regulatory considerations for risk assessment of GMMs before field release. Figure 1 gives a schematic overview of the use of biomarkers/bioreporters in GMM releases. The use of any type of marker/reporter is seen by many as being essential in assessing the environmental fate of release organisms.

Each chapter of this book is authored by at least one participant of the MAREP Concerted Action of Scientists. MAREP is an acronym for "Marker/ Reporter genes in Microbial Ecology" and is comprised of 26 scientists from 11 different countries. The MAREP Concerted Action is funded by the European Commission, Directorate-General XII, and addresses issues related to the use of marker and reporter genes in microbial ecology research. This is of particular importance in relation to the precise monitoring of GMM presence, persistence and metabolic activity in the environment. More information about the MAREP Concerted Action can be found in the following Web site: http://www.biokemi.su.se/marep/marep.html.

We intend this book to be of relevance for all those concerned with studies of the environmental fate of genetically modified or unmodified microorganisms. In particular, this volume will be of value to researchers developing organisms intended for release, and to representatives of regulatory agencies concerned with guiding experimental or commercial applications. This volume provides an up-to-date collection of data on the development, use and assessment of biomarkers and bioreporters for the study of bacterial function in the natural environment.

We would like to extend our thanks to all the authors for providing the necessary text for this publication, and for the patience and understanding they have shown during the editing process. We would also like to thank the editorial staff for their support.

Janet K. Jansson Jan Dirk van Elsas Mark J. Bailey

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