Andréa Wiktor Gabriel

Sophie Madelrieux

Philippe Lescoat

A review of socio-economic metabolism representations and their links to action: cases in agri-food studies

Keywords: research-management interface, participative research, metabolic networks, epistemology, pragmatism 1

come

A review of socio-economic metabolism representations

and their links to action: Cases in agri-food studies

1 Introduction: socio-economic metabolism and action Socio-economic metabolism is seen as a paradigm for studying the biophysical basis of our societies. It " constitutes the self-reproduction and evolution of the biophysical structures of human society. It comprises those biophysical transformation processes, distribution processes, and flows, which are controlled by humans for their purposes [and] the biophysical structures of society" [START_REF] Pauliuk | Socioeconomic Metabolism as Paradigm for Studying the Biophysical Basis of Human Societies[END_REF]. The notion of SEM has evolved over time and the scope of the term differs among scholars. Socio-metabolic studies translate into a wide diversity of operational concepts, such as industrial [START_REF] Frosch | Strategies for Manufacturing[END_REF]), society's [START_REF] Fischer | Society's Metabolism[END_REF], urban [START_REF] Barles | The Main Characteristics of Urban Socio-Ecological Trajectories: Paris (France) from the 18th to the 20th Century[END_REF] and socio-economic metabolism [START_REF] Pauliuk | Socioeconomic Metabolism as Paradigm for Studying the Biophysical Basis of Human Societies[END_REF]). In the remainder of the text, we will use "socio-economic metabolism" (SEM) to refer to the paradigm in general.

Several bibliographical reviews concerning SEM studies have recently been published.

They include a comparison of two frameworks (Gerber et Metabolism is indeed a tool for management. According to [START_REF] Howard | Environment, Power, and Society[END_REF], ecologists should use metabolism as a tool to share their analysis of reality with society and, finally, to position themselves as the actual managers of the interactions between society and the environment. The final goal is to "manage" the human system with actions based on ecological laws (Madison 1997). Social metabolism is seen as a tool for socio-ecological transition [START_REF] Fischer-Kowalski | Conceptualizing, Observing, and Influencing Social-Ecological Transitions[END_REF]), ecological intensification (González de Molina et al. 2017) or sustainability [START_REF] Haberl | Contributions of Sociometabolic Research to Sustainability Science[END_REF].

While action plays an important role in SEM studies, it is rarely addressed as such in the reviews. What are researchers normative and operational objectives? What do they stand for and how can they be achieved? How does action relate to choices of metabolic representation? These questions are central if we want to address the environmental, social and political challenges revealed by metabolic representations.

In agri-food studies SEM is increasingly present in agri-food studies. Agri-food systems play a major role in the biophysical basis of our societies and strongly contribute to society's SEM in terms of material stocks and flows (Krausmann et al. 2009). Material flows and, in particular, flows of biomass or living matter are the subject of new challenges with the development of the bioeconomy (Vivien et al. 2019). Agriculture is the main producer of biomass in human economies by far, reaching 89 % in total mass. The biomass of humans and livestock surpass that of wild mammals by a ratio of 20 [START_REF] Yinon | The Biomass Distribution on Earth[END_REF]).

Development of SEM in the industrial age includes a metabolic rift between the city and the countryside [START_REF] Bellamy | Marx's Ecology: Materialism and Nature[END_REF] as well as agricultural sector specialization [START_REF] Lemaire | Integrated Crop-Livestock Systems: Strategies to Achieve Synergy between Agricultural Production and Environmental Quality[END_REF]. It translates into an environmental footprint [START_REF] Courtonne | Environmental Pressures Embodied in the French Cereals Supply Chain[END_REF]) such as nitrogen pollution [START_REF] Bellarby | Strategies for Sustainable Nutrient Management: Insights from a Mixed Natural and Social Science Analysis of Chinese Crop Production Systems[END_REF] or depletion of organic matter in the soil [START_REF] Andrieu | From Farm Scale Synergies to Village Scale Trade-Offs: Cereal Crop Residues Use in an Agro-Pastoral System of the Sudanian Zone of Burkina Faso[END_REF]).

The unsustainable nature of the ongoing SEM is unanimously recognized and a consensus exists on the urgency to transform it [START_REF] Haberl | Contributions of Sociometabolic Research to Sustainability Science[END_REF].

Goals and article map

We propose a review of the literature based on SEM representation and action in agri-food systems. This includes agricultural production, food systems and their associated components such as inputs and waste flows. Several steps are followed. Firstly in section 2, methods are chosen to isolate the relevant literature, describe SEM representation and the researchers' links to action. Thanks to these tools, section 3 is an attempt to characterize schools of thought regarding SEM and action. Section 4 presents some limits of the review and discusses the fact that representations and action are linked: studies of metabolism are not neutral and "schools of thought" are also "schools for action". We conclude with a proposal of epistemological and methodological choices for SEM, anchored in pragmatism.

Methodology 2.1 Identification of literature dealing with SEM through keywords

A systematic exploration of the literature was carried out to identify articles that deal with SEM in agri-food systems. We looked for papers that focus on the biophysical structures of human societies, seen in terms of human-controlled processes of transformation and distribution. We interpreted the "human society" criteria in a restrictive way: the flows must have a social aspect, e.g., taking place on a scale larger than that of the farm, or involving a collective process.

Searches in the scientific literature were performed using the Web of Science, with three sets of keywords: those referring to (1) metabolic processes (e.g., metabo*1 ; material; flow; biomass; circulation); (2) agri-food systems (e.g., agri*; farm*; agrarian; agroecosystem); and (3) social (e.g., social, societ*, collective, politic* ). Results of the search cover the period from 2000-2019. 738 articles featured at least one keyword for each category. After excluding off-topic articles, 259 publications remained. While almost no authors explicitly used the concept of SEM, we ensured that all three criteria were met in the articles' abstracts.

A total of 89 articles remained. On this basis, 13 additional articles were identified via the snowball effect. The total number of articles analyzed was 102.

Literature description and classification

The articles are described according to their SEM representation and their links with action (Table 1). Theoretical articles were used to describe the school of thought, and case studies to illustrate methodological choices. Complementary articles, not described in the review, and not specialized in agri-food, were used to describe the schools of thought. These articles are mainly theoretical or seminal. Each school is then described in a synthetic way, according to how the metabolism is represented, as well as its relationship to action. Two to three case studies are proposed as an illustrative example for each school.

Based only on the SEM representation, we identified eight "schools of thought" in which the various articles are classified. When various but similar names coexisted (e.g., industrial

Representation (a) Stocks flows and funds

Stocks and flows; economic funds and flows; ecological funds and flows; humans and non-humans as funds and flows

(b) Scales and levels

Large-scale spaces; middle or small scales; physical black boxes; individual companies or sectors;

(c) Socio-economic context

Historical material transitions (Marxist) ; social performance analysis ; power structure analysis; local stakeholder analysis; actor-networks

Action (d) Claimed research goals

Limit the size of the metabolism; change the system; relocalize the economy; foster economic performance; limit environmental impact; close the loop ; preserve the ecological and economical funds; develop an ecological-process-based and socially fair food system; enhance diverse and locallybased sustainability programs

(e) Researchmanagement interface

Trickle-down; user-push; transfer-and-translate; researchwith-management

(f) Tools

Transition analysis; critical analysis; stakeholder analysis; management tools; post-normal-science-based tools; extended description (g) Actors and partners Leaders of countries; civil society; local authorities and stakeholders; companies; trade associations ; farmers and civil society; various stakeholders.

Table 1: List of literature classification criteria according to the type of SEM representation and links with action ecology, industrial clusters and industrial symbiosis), a compromise was made (e.g., industrial ecology and symbiosis). For the sake of simplicity, only the most prominent and contrasting frameworks were chosen. (a) We followed Georgescu-Roegen's (1971) proposal to represent human-mediated metabolic processes using three distinct categories: stocks, flows and funds. A stock is what is present in the system at a given moment of time. A flow represents change: it is usually used for representing an input or an output of a given process. Funds are durable entities, which are the "active agents of the process", while flows are "used or acted upon by the agents" (Georgescu-Roegen 1971). A diversity of entities can be taken into account. These include economic or ecological processes, human and non-human agents.

(b) Living systems present parallel levels of organization on different scales and levels.

Spatial scales (e.g., region, state, global, etc.) and functional levels (humans are made of organs, cells, molecules, etc., which at the same time are part of a household, part of a community, part of a country) [START_REF] Giampietro | Multi-Scale Integrated Analysis of Agroecosystems[END_REF]). For each study, we attempted to describe the scales and levels that are taken into account in metabolic representations. For scales, these include: large-scale spaces, middle or small scales; for levels: physical black boxes, individual companies or sectors.

(c) The biophysical basis of our societies is rarely described alone. It is regularly integrated into a socio-economic context. Its analysis encompasses the diversity of the social sciences: historical, sociological, political, etc. The main types of analyses used were derived from the reading of the corpus. These include historical material transitions, social performance analysis, (Marxist) power structure analysis, local stakeholder analysis, actor-networks.

Action: goals, interfaces, tools and partners

The links between SEM representation and action are analyzed on the basis of four criteria:

(d) problem definition and the researcher's goal; (e) type of research-management interface;

(f) type of analysis or tools used; and (g) the chosen actors and partners.

(d) Problem definition and the researcher's goal are deduced from what the author states in the article. These objectives can be expressed explicitly or make reference to theoretical articles, e.g., limit the size of the metabolism, change the system, re-localize the economy, foster economic performance and limit environmental impact.

(e) The types of research-management interfaces are characterized using the typology of [START_REF] Gosselin | Ecological Research and Environmental Management: We Need Different Interfaces Based on Different Knowledge Types[END_REF], which distinguishes four types of interfaces between research and management: trickle-down, transfer-and-translate, user-push and research-within-management (Fig. 1).

1-In trickle down interfaces, knowledge is generated with no direct link to management.

Researchers produce research, independently of topics of interest to users. Users can adopt it if they wish, but the researcher makes no effort in that direction.

2-In transfer-and-translate interfaces, scientists make an effort to transfer their results, while managers translate them into coherent management practices (e.g., extension officers at the interface between scientific knowledge and farmers).

3-In user-push interfaces, the users commission research on topics they are interested in.

Managers may ask researchers to produce knowledge that will inform their future management.

4-Research-within-management is based on strong interactions through bidirectional flows of knowledge. Researchers and managers work together, pushing and pulling knowledge to define research questions and to conduct research relevant to their mutual skills and needs.

(f) The type of analysis or tools provided by the researcher for the action is described.

It includes transition analysis, critical analysis, stakeholder analysis, data and management tools, post-normal-science-based analysis and exchanges and extended descriptions.

(g) The actors described as potential agents for change as well as the ones who are partners with whom the researchers collaborate are identified. These include leaders of countries, civil society, local authorities and stakeholders, companies, farmers and civil society, as well as various stakeholders. Each of these types contains several schools of thought. The way each school represents the metabolism and the relationship to action is described below (Sections 3.1 to 3.3), and summarized in Table 2.

Space and compartment-based representations

Space and compartment-based representations were developed, in particular, by [START_REF] Fischer-Kowalski | Social Metabolism: A Metric for Biophysical Growth and Degrowth[END_REF]. These types of approaches consider society as a set of black boxes within which flows of matter and energy pass and are disposed of (Gomiero 2017).

We identified three main schools of thought: (1) social ecology; (2) metabolic rift and Marxist ecology; and (3) urban and territorial ecology.

Social ecology

In this school of thought, articles are generally far-reaching descriptions of metabolic changes and dynamic studies, taking the evolution of agri-food systems into account. They focus on large-scale spaces like countries or regions, and link them to broad historical analyses such as long-term socio-ecological transitions. Flows represent materials (e.g., biomass production and appropriation of the net primary production of a country [START_REF] Kohlheb | Land Use Change, Biomass Production and HANPP: The Case of Hungary 1961-2005[END_REF])), energy (e.g., the systemic account of a nation's carbon budget, comprising socioeconomic as well as ecological carbon flows in a historic time series [START_REF] Erb | How a Socio-Ecological Metabolism Approach Can Help to Advance Our Understanding of Changes in Land-Use Intensity[END_REF][START_REF] Cusso | Social Metabolism in an Agrarian Region of Catalonia (Spain) in 1860-1870: Flows, Energy Balance and Land Use[END_REF])). Stocks consist of human and non-human populations such as livestock, infrastructure or land use [START_REF] Fischer-Kowalski | Conceptualizing, Observing, and Influencing Social-Ecological Transitions[END_REF]). These "black box" representations do not describe agents or "funds" as such [START_REF] Frankova | Socio-Metabolic Perspectives on the Sustainability of Local Food Systems[END_REF]. Instead, authors take an holistic or whole system approach, for example describing the effect of a whole country's metabolism in terms of environmental

School of thought (number of articles)

Representation

Action Articles Space and compartment-based representations [START_REF] Fischer-Kowalski | Conceptualizing, Observing, and Influencing Social-Ecological Transitions[END_REF] Social ecology [START_REF] Brunori | Are Local Food Chains More Sustainable than Global Food Chains? Considerations for Assessment[END_REF] Stocks and flows at large scale spaces or between physical black boxes analyzed through historical material transitions

Limit the size of the metabolism through trickle-down and transition analysis directed at leaders of countries . The text follow the same pattern, with the criteria described in order. In the column "Representation" : (a) funds and flows, (b) their scale, (c) analyzed through their socio-economiccontext. In the column "Action": (d) Goal, (e) the research-management interface, (f) the tools with (g) the actors.

impacts or pollution [START_REF] Gueldner | Nutrient Recycling and Soil Fertility Management in the Course of the Industrial Transition of Traditional, Organic Agriculture: The Case of Bruck Estate, 1787-1906[END_REF], or an analysis of the transition between different metabolic regimes [START_REF] Soto | The Social Metabolism of Biomass in Spain, 1900-2008: From Food to Feed-Oriented Changes in the Agro-Ecosystems[END_REF]). On this scale, it is not easy to deal with actors and their deliberate efforts. The agents in charge of governance are out of scope: socio-economic context is often disconnected from the description of stocks and flows. Researchers aim for the sustainability of resource use or the resilience of societies. They are interested in the size of the metabolism and, more or less explicitly, seek to reduce its size to a sustainable level.

Research-management relies on a trickle-down interface: stakeholders are rarely involved and they almost never commission the research to promote change themselves [START_REF] Fischer-Kowalski | Conceptualizing, Observing, and Influencing Social-Ecological Transitions[END_REF]). Researchers promote change through knowledge, which is provided in a top-down way without taking the needs of any specific agents into account or trying to translate this knowledge in terms more suitable for policy making. When described, the potential actors are often public actors (e.g., national governments or international institutions). Facts come with no insight into what should be done, e.g., how global governance of flow could be implemented in the real world.

Metabolic rift and Marxist ecology

These articles explore a range of metabolism-related themes inspired by Marx's theories. The focus is on the same scales as those of social ecology (e.g., stocks and flows in a region-wide agricultural sector, long-term transformations), although the quantitative analysis of flows is often more succinct. What could act on metabolism and, consequently, on the capitalist system, is rarely described. Marxist descriptions of metabolism are, according to Georgescu-Roegen (1971), of the stocks-and-flows type. Funds or agents' abilities to change the system are not central. When they are mentioned, it is recalled that their capacity to change the system is limited [START_REF] Gunderson | The Metabolic Rifts of Livestock Agribusiness[END_REF]). Authors rely explicitly on Marxist concepts such as the metabolic rift. This concept refers to the rupture between humanity and nature and, extensively, between the city and the countryside [START_REF] Bellamy | Marx's Ecology: Materialism and Nature[END_REF][START_REF] Moore | Transcending the Metabolic Rift: A Theory of Crises in the Capitalist World-Ecology[END_REF]). The metabolic rift perspective has been since used in a large variety of case studies. Performance is measured in terms of rifts in soil nutrient cycles or in terms of structural changes in relation to global carbon, nitrogen and water cycling [START_REF] Gunderson | The Metabolic Rifts of Livestock Agribusiness[END_REF]). The metabolic rift presents strong and multiple connections with other Marxist analyses, refering to the socio-economic context, such as these concerning capital or labor, and emphasizing power structures and exploitation [START_REF] Bellamy | Marx's Ecology: Materialism and Nature[END_REF][START_REF] Gunderson | The Metabolic Rifts of Livestock Agribusiness[END_REF]).

The claimed goal is to change the system towards more social or environmental justice.

To propose an alternative and support their arguments, several authors rely on comparisons with non-capitalist economies, e.g., Cuba [START_REF] Clausen | Metabolic Rifts and Restoration: Agricultural Crises and the Potential[END_REF]). The research-management interface is mainly based on a trickle-down model, and rely on critical analysis to generate change. Links with ongoing actions, when mentioned, associate social metabolism with environmental justice struggles lead by the civil society (Martinez-Alier et al. 2010).

Urban and territorial ecology

Urban and territorial ecology describe flows at local scales (e.g., territories or cities). At these scales, local authorities, economic agents, populations and living beings are described as active agents and funds. This gives rise to stakeholder analyses, sometimes inscribed in a broader historical context. [START_REF] Barles | Urban Metabolism and River Systems: An Historical Perspective -Paris and the Seine, 1790-1970[END_REF] analyzed metabolic interaction between Paris and the Seine during the industrial era. Funds at the scale of a territory are extensively described (administrative authorities, city-scale policies). Other levels are also described: lower ones (individual companies, civil society), or higher ones (changes in the technological or economic landscape, state-driven planning). [START_REF] Buclet | Essai d'écologie territoriale: l'exemple d'Aussois en Savoie[END_REF] described different subsystems within a territory (agri-food system, wealth creation system, etc.) and explained the dynamics in the light of global factors and individual institutions. The goal is to produce analyses and representations at the scales of local stakeholders in order to reach a sustainable development of the territory e.g., through flow relocalization. These studies regularly involve a transfer-and-translate interface with the local authorities, to whom data and management tools are provided. The terms and concepts used are often very close to those used by administrative agents, making exchanges easier.

Economic agent-based representations

Articles in this type of SEM representation describe metabolism in terms of material flows between individual economic agents. We distinguish two forms: (1) a linear representation, the supply chain, where actors are distributed according to their place in the production process, "from cradle to grave", or from resource extraction to waste management; and [START_REF] Alfaro | Applying Industrial Symbiosis to Smallholder Farms: Modeling a Case Study in Liberia, West Africa[END_REF] an ideal form, the perfect circle, in which there is no external resource or waste: the industrial symbiosis.

Supply-chains-based metabolism

These studies are concerned with the organizations involved during the production process of a product, from the extraction of resources to the delivery of the finished product to a consumer (or beyond). They take a functional level standpoint and distinguish processes according to the role played in the chain, e.g., production, transformation or distribution. Studies exist at all levels (local individual companies to global supply chains), and characterize metabolism through life-cycle analysis (LCA) or material flow analysis (MFA).

Socio-economic context is integrated through economic and stakeholder analysis such as economically-extended MFA [START_REF] Kytzia | Economically Extended-MFA: A Material Flow Approach for a Better Understanding of Food Production Chain[END_REF]), social networks [START_REF] Xu | Understanding the Mechanism of Food Waste Management by Using Stakeholder Analysis and Social Network Model: An Industrial Ecology Perspective[END_REF]) or scenario building [START_REF] Kulak | Eco-Efficiency Improvement by Using Integrative Design and Life Cycle Assessment. The Case Study of Alternative Bread Supply Chains in France[END_REF]). The goal is to help economic agents to limit environmental impacts (e.g., resource extraction, waste, pollution or carbon footprints), while maintaining or fostering economic performance. Most of the studies are related to a "transfer-and-translate" interface. The results are intended to be directly discussed with the decision makers, mainly the companies, but also trade or sector associations. The fact that the results or the tools can be directly mobilized by the actors is regularly a top priority. For example, Kulak et al.

(2016) analyzed the bread supply chain using LCA to generate scenarios with experts during a collaborative design workshop, and then discussed the scenarios with farmers on a feedback loop basis. Blengini and Busto (2009) discussed the environmental impacts of alternative rice production systems using LCA and proposed their results as a tool for communication between suppliers and their customers.

Industrial ecology and symbiosis

2017).

The goal of these approaches is to "close the loop" of materials and substances. Natural ecosystems are proposed as models for industrial activities: "Our industrial system [would] behave like an ecosystem, where the wastes of a species [are] a resource to another species.

The outputs of an industry [would] be the inputs of another, thus reducing use of raw materials and pollution" [START_REF] Frosch | Strategies for Manufacturing[END_REF]). The paradigmatic vision of sustainable industrial systems is characterized by minimized physical exchanges with the environment [START_REF] Wassenaar | Reconsidering Industrial Metabolism: From Analogy to Denoting Actuality[END_REF].

Researchers regularly work closely with companies and operators. They are involved in the choice of research directions and are the subject of particular attention in terms of translation of the results. Resource-use optimization is often considered to be synergistic with economic performance (or profit making). Researchers seek to provide data or tools that allow companies to better manage interactions within their economic ecosystem. Most of these studies rely on user-push or transfer-and-translate interfaces. In some cases involving long-term relationships, researchers take part in a research-within-management interface, aimed at building a planned industrial symbiosis [START_REF] Iacondini | Feasibility of Industrial Symbiosis in Italy as an Opportunity for Economic Development: Critical Success Factor Analysis, Impact and Constrains of the Specific Italian Regulations[END_REF]).

Multi-faceted and composite representations

The articles in this type of SEM representation do not describe agri-food systems in terms of large-scale stocks and flows, nor do they propose an economic-agent-based funds and flows analysis. We present these "atypical" multi-faceted and composite representations of metabolism through three examples: (1) multi-scale analysis of agroecosystems; (2) agroecology within food systems and landscapes; and (3) pragmatic sociology and earthbound ecology.

Multi-scale intergated analysis of agroecosystems

This framwork is more broadly termed as Multi-Scale Integrated Analysis of Societal and Ecosystem Metabolism (MuSIASEM). It provides analysis of agroecosystems in terms of material and energy flows as well as biophysical and socio-economic funds. It provides theory and operational tools for characterization across multiple hierarchical levels of the performance of socio-economic activities [START_REF] Giampietro | Multi-Scale Integrated Analysis of Societal and Ecosystem Metabolism (MuSIASEM): Theoretical Concepts and Basic Rationale[END_REF]). Serrano-Tovar et al. (2014) characterize the socioeconomic activities by a series of quantitative indicators at different scales (individual process, household, community). Humans workforce and the land are represented as funds, while flows are both material and economic. The main goal is the assessment of technical performance and better consideration of the biophysical constraints at the basis of economic activity. The sustainability is assessed in terms of viability (resources used and waste produced at rates compatible with those of the biophysical environment), feasiblity (human labour available), and desirability. The aim is to preserve the economic and ecological funds which ensure the metabolism works. In this sense, MuSIASEM is in direct continuity with Georgescu-Roegen's approaches.

When dealing with action, theoretical articles of MuSIASEM often refer to post-normal sciences. This method is intended to be a response to situations in which "the facts [are] uncertain, values in dispute, stakes high and decisions urgent" [START_REF] Funtowicz | Science for the Post Normal Age[END_REF] This negotiation and participatory part, although described as essential in theory (Giampietro 2004), is nevertheless not described in every case study. Many studies follow similar methods without referring to MuSIASEM in the strict sense, nor following a post-normal approach. Gonzalez-Acevedo et al. (2016) compare different coffee systems according to ecological economic indicators (e.g., economic, energy performance and self-sufficiency), on the scale of households and society. The results are provided to coffee traders through a trickle-down interface.

Agroecology within food systems and landscapes

Agroecology is "the integrative study of the ecology of the entire food system" [START_REF] Francis | Agroecology: The Ecology of Food Systems[END_REF]. It emphasizes the interrelatedness of all agroecosystem components and the complex dynamics of ecological processes [START_REF] Altieri | Agroecology : The Science of Natural Resource Management for Poor Farmers in Marginal Environments[END_REF]): agronomic and ecological analyses are combined with social or cultural aspects. Metabolic processes are given special attention [START_REF] González | Agroecology and Ecological Intensification. A Discussion from a Metabolic Point of View[END_REF]). These processes include nutrient cycling, crop-livestock interactions [START_REF] Bonaudo | Agroecological Principles for the Redesign of Integrated Crop -Livestock Systems[END_REF][START_REF] Martin | Crop-Livestock Integration beyond the Farm Level: A Review[END_REF]) and material flows in food systems [START_REF] Francis | Agroecology: The Ecology of Food Systems[END_REF][START_REF] Vaarst | Exploring the Concept of Agroecological Food Systems in a City-Region Context[END_REF]). Whereas traditionally focused on farm and plot scale, the landscape, community and multi-scale approaches are receiving increasingly more attention. The description of the socio-economic context is based on analyses of power structures.

Science and action are considered together. Agroecology provides the basic ecological principles for how to study, design and manage agroecosystems [START_REF] Altieri | Agroecology : The Science of Natural Resource Management for Poor Farmers in Marginal Environments[END_REF]. It is both a science, an agricultural practice and a political movement [START_REF] Wezel | Agroecology as a Science, a Movement and a Practice. A Review[END_REF]). Researchers claim normativity as their goal. They aim at a more sustainable agricultural system based on a strong dependence on ecological processes or services as well as on social justice. Action is considered broadly and involves civil society, NGOs, academics, local authorities, etc. Stakeholder knowledge and perception is analyzed in relation to these programs. The results provide areas for management improvement.

Pragmatic sociology and earthbound ecology

In agro-food studies, this school of thought brings together constructivists approaches that have challenged previous understandings and include frameworks such as the actor-networktheory [START_REF] Goodman | Ontology Matters: The Relational Materiality of Nature and Agro-Food Studies[END_REF] They focuses attention on hybridity and the role of heterogeneous associations in complex networks [START_REF] Goodman | Ontology Matters: The Relational Materiality of Nature and Agro-Food Studies[END_REF], and seek to understand what is happening in the process of building and stabilizing networks. Both humans and non-humans entities are considered as active agents [START_REF] Callon | Techno-Economic Networks and Irreversibility[END_REF]). The researchers follow the actors in the situations they encounter, and provide qualitative descriptions of links. In agri-food studies, this implies following material flows through their transformations, in markets and technical devices. Representations describe composite consisting of heterogeneous elements including humans, materials and technical devices that flexibly adjust to one another and act collectively (C ¸alışkan et al.

2010

). No scales are explicitly described. [START_REF] Akrich | Sociologie de la traduction: textes fondateurs[END_REF] studied the process of network building around cane straw as a source of energy for households. The evolution of the straw flow is followed, and its transformations are analyzed in connection with other agents (technological tools, institutions, etc). The straw flow is not only a flow, in Georgescu-Roegen's terms, but also a real agent, that actively transforms other agents around it. Wegerif et al. ( 2016) use an ethnomethodological approach to follow the agents implied in the food chain of a town, and trace interactions between them, highlighting transformations of food, people and ideas throughout the process.

Socio-economic aspects are not considered as context, but rather considered as an integral part of the metabolism. These are described jointly as actor-networks.

The goal of pragmatic sociology is not normative, but procedural: it intends to bring attention to the network of ties that binds us to all life forms. These attachments sometimes described as "down-to-Earth", "earthbound" or "terrestrial" form the basis for a new definition of ecology [START_REF] Latour | Facing Gaia: Eight Lectures on the New Climatic Regime[END_REF]

Discussion

Limits and weaknesses

Our literature survey did not capture all the diversity of metabolic approaches. The keyword approach has made it possible to identify a variety of approaches from distant disciplines. However, it excluded relevant work only for vocabulary reasons. This is problematic since our field of study presents strong conceptual variability. Harvesting additional articles via the snowball effect has at least partially filled this gap. On the other hand, the method underestimates the non-English documents or books that were recovered only indirectly. The stock/flow/fund framework proposed by Georgescu-Roegen (1971) is sometimes difficult to operationalize. In practice, it is not always easy to distinguish whether an author refers to stocks or funds. Concerning socio-economic analyses, criteria such as quantitative/qualitative or micro/macro would also have led to a different classification. For science and technics scholars, the relationship to action is considered as a process of translations where representations and actors mutually transform each other through a dynamic process [START_REF] Callon | Some Elements of a Sociology of Translation: Domestication of the Scallops and the Fishermen of St Brieuc Bay[END_REF]. This type of interpretation would further require qualitative description work, which would be difficult to integrate into a simple analysis grid.

The schools of thought are constructed according to our reading of SEM representations and links with action that we perceived in the articles. The authors of the papers do not always claim to be from the schools in which they are classified. For example, the article by Mol et al. ( 2006) is mainly based on descriptions characteristic of the school of thought "Pragmatic sociology and earthbound ecology". However, these authors claim to rely partially on industrial ecology, without endorsing its goals.

Representation and action are deeply intertwined

Our work showed a diversity of schools, described in terms of the way they deal with SEM (representation and action) (Fig. 2). The way in which representations and action are intertwined is discussed by type of representation:

In space and compartment-based representations

In space and compartment-based representations scales usually correspond to administrative scales, e.g., countries or regions [START_REF] Fischer-Kowalski | Conceptualizing, Observing, and Influencing Social-Ecological Transitions[END_REF], which are usually "blackboxed". Action is not explicitly the main goal of these studies: researchers outline the problem and describe some of the quantities to be dealt with. The goal is normative and global, whether it is to change the economic system (Marxist ecology), or simply to drastically reduce its size (social ecology). Stakeholders are rarely involved and they are usually mobilized in a trickle-down interface. This contrasts with studies focused on smaller spatial scales such as urban or territorial ecology. They focus on smaller spatial scales and their goal is more agent-centered (e.g., sustainability of a given territory in urban and territorial ecology). Biophysical constraints are expressed at a territorial scale in a way favorable to their being taken into account by institutions: e.g., an Austrian-wide description is well suited to the Austrian authorities. Furthermore, when the metabolism is described at the scale of a given region, the administrative authorities of that region are regularly mentioned as partners. Policymakers and beyond them, governments or states, are assumed to be interested in handing collective problems such as the excessive size of SEM [START_REF] Görg | Challenges for Social-Ecological Transformations: Contributions from Social and Political Ecology[END_REF]).

In economic agent-based representations

In economic agent-based representations authors explicitly claim their interest in action. The operational goal is agent-centered: economic, technical or environmental performance (e.g., closing the loop) is considered to be synergistic with the economic interest of companies.

Representation and actions are closely intertwined and this is reflected, in particular, by the place given to economic actors. Economic agents are given a central role since businesses are seen as the main actors able to handle change and technological innovation, and these are seen as essential for environmental improvement (Ayres et al. 2002). They are considered as economic funds, analyzed as stakeholders, and considered as partners in research-management interfaces. Agents that are described as funds in SEM representations are the same as those that are considered as potential partners through user-push or transfer-and-translate interfaces.

In multi-faceted and composite representations

Multi-faceted and composite representations offer descriptions that are neither centered mainly on economic actors, nor on predetermined spaces or compartments. Multi-scale analysis of agroecosystems, agroecology or pragmatic sociology do this in different ways: they question scales, challenge the science/politics divide or integrate humans and non-humans, respectively. All rely on a representation of multiple funds and actors at different scales or in a non-scalar way. Each of these approaches proposes a renewed way of dealing with action.

All these approaches explicitly consider that the relationship to action must be taken into account in scientific representations (by post-normal science, by taking into account indigenous knowledge, or by the performativity of descriptions, respectively). The authors insist on the importance of taking multiple agents into account when generating representations (choice of scales, entities to be represented), and discussing them through collective deliberation in a research-within-management interface.

The theoretical and practical limits of schools of thought

Each school of thought chooses to open different black boxes and focuses only on a part of the system. Representations focused on specific scales or actors favor certain (agricultural) models over others, e.g., a large-scale SEM representation would favor the dominant systems, masking a variety of alternative models. The fact that companies are described as central in industrial ecology helps to rule out any alternative model. Historians' work shows that this legitimization of industries through metabolic representations has roots that go back to the beginning of industrialization in the 19th century [START_REF] Fressoz | La Main Invisible A-t-Elle Le Pouce Vert ?: Les Faux-Semblants de "l'écologie Industrielle "Au Xixe Siècle[END_REF]). Thus, each school defines different incommensurable visions of what a sustainable SEM should be and how it should be changed: schools of thought are also "schools for change". However, these positions are not definitive: there are not only variations within schools of thought, but also exchanges between them.

Practical cases diverge from theoretical claims

In some situations there is a discrepancy between the positions defended in the theoretical articles and what is actually described in the case studies. For example, MuSIASEM follows the proposals of post-normal science and states that researchers should take part in social negotiations and co-evaluate the results with stakeholders with a communication on uncertainties [START_REF] Giampietro | Complexity and Scales: The Challenge[END_REF]). However, these recommendations rarely appear in case studies.

Agroecology claims that science and politics cannot be separated, and farmers, researchers and civil society must be associated in the construction of knowledge, practices and social struggles [START_REF] Altieri | The Agroecological Revolution in Latin America: Rescuing Nature, Ensuring Food Sovereignty and Empowering Peasants[END_REF]). Nonetheless, the case studies do not always show such a transdisciplinary posture. This difference generates dissensions and different competing narratives (Rivera-Ferre 2018).

Schools of thought transform in relation to each other

The different schools of thought interact and define themselves in relation to each other. For example, agroecology presents itself as being opposed to input substitution like that proposed by industrial ecology [START_REF] Peter | Agroecology versus Input Substitution: A Fundamental Contradiction of Sustainable Agriculture[END_REF], and opposes the idea that sustainability could be attained through a mono-supply chain approach [START_REF] Francis | Agroecology: The Ecology of Food Systems[END_REF]. MuSIASEM proposes to focus on funds, as opposed to the stock and flow descriptions found in social ecology, and proposes decision-making based on social negotiation as opposed to the reductionist computer-based optimization found in industrial ecology [START_REF] Giampetro | Complexity and Scales: The Challenge for Integrated Assessment[END_REF]. Territorial ecology considers itself different from industrial ecology seen as focusing exclusively on resource-use optimization or on industrial societies, and leaves room for non-material flows in its analysis [START_REF] Buclet | Essai d'écologie territoriale: l'exemple d'Aussois en Savoie[END_REF].

Epistemological and methodological consequences

SEM studies find themselves in a balance between two epistemologies, positive and practical.

While the majority of researchers aim to a certain extent to produce positive or neutral results [START_REF] Pauliuk | Socioeconomic Metabolism as Paradigm for Studying the Biophysical Basis of Human Societies[END_REF], many SEM studies have a practical focus, judging their results by their usefulness in influencing reality. This raises the question of unstructured methodological pluralism [START_REF] Spash | The Shallow or the Deep Ecological Economics Movement?[END_REF]. In order to "unite the community", encourage "fruitful exchanges" or favor "change", "action", a certain number of authors have proposed definitions, epistemological or methodological positions and a set of attributes to be preserved in metabolic representations and in SEM in particular (

The issue of pluralism

How do pragmatists approach the issue of pluralism, which is of interest to us here? William James describes two opposing philosophical positions: monists and pluralists. For the monists, the world has an intrinsic and absolute unity. For pluralists, it is disjointed, and there are as many worlds as there are points of view. Pragmatists refuse to decide between these two extremes, seeking instead a mediating way. Wondering about the One and the Many from a pragmatic perspective means answering the following question: "Granting the oneness to exist, what facts will be different in consequence? [...] What is the practical value of the oneness for us?" (James 1907).

A relational ontology

The pragmatic way consists in investigating what unites different elements, one by one.

The world is one in the measure of the sum of the connections that we can discover in the experience. Oneness is described in the terms of links or relationship, i.e in a relational ontology.

Multiple representations

Nevertheless, the criteria for judging links are innumerable, the experience is multiple [START_REF] James | Pragmatism[END_REF]). The diversity of criteria for satisfaction (or assessment) implies that there is necessarily a plurality of values, and therefore a plurality of possible representations.

Collective action and deliberation

The world is also multiple in the sense that the future of the world is indeterminate, it holds possibilities that cannot be predetermined. It always has something incomplete, something in the making. This means that the action and perspective of each human being counts.

Pragmatism attaches great importance to collective action [START_REF] Madelrieux | Le pragmatisme de William James (Presentation)[END_REF], and especially deliberative and democratic processes that respect this pluralism (Rorty 1999).

Socio-ecological metabolism from a pragmatic perspective

Our results show that the practical consequences of pragmatic thinking are already and variously taken into account by many schools of thought: 4.4.3.1 An empirical approach: metabolism as a reality rather than an analogy Flows in industrial systems used to be compared to flows and processes internal to an organism. The reference to physiology is less used today, and it is the reference to ecology, the science which actually describes the movement of materials and energy through living communities, that is preferred [START_REF] Pauliuk | Socioeconomic Metabolism as Paradigm for Studying the Biophysical Basis of Human Societies[END_REF][START_REF] Wassenaar | Reconsidering Industrial Metabolism: From Analogy to Denoting Actuality[END_REF]. Social ecology proposes representations which describes social and natural metabolisms in the same ecological terms [START_REF] Fischer-Kowalski | Conceptualizing, Observing, and Influencing Social-Ecological Transitions[END_REF][START_REF] Kohlheb | Land Use Change, Biomass Production and HANPP: The Case of Hungary 1961-2005[END_REF]). MuSIASEM uses the funds/flows framework to investigate both human and non-human-made funds in economic processes. Pragmatic sociology and "earthbound" refer, often explicitly, to ecology.

A relational ontology through metabolic networks

Most schools refer more and more explicitly to a relational ontology. In industrial ecology and supply chains, some authors refer to "metabolic network", i.e., "a subset of a complex system of interconnected transformative processes across all scales of life", or rely on social and material network analysis [START_REF] Schiller | Analyzing Networks in Industrial Ecology -a Review of Social-Material Network Analyses[END_REF][START_REF] Wassenaar | Reconsidering Industrial Metabolism: From Analogy to Denoting Actuality[END_REF]. In Marxist ecology, nonbinary concepts such as the "metabolic shift" are recently preferred to the historic "metabolic rift", in order to focus on the transformations, rather than on the rupture between two ideal systems (Moore 2017). In pragmatic sociology, it means abandoning levels or scales and focusing instead on intensifications and extensions of entangled dependencies (Conway 2016).

Multiple representations: multiple scales, agents and values

Pluralism in terms of representations is widely shared: the SEM does not prescribe a specific level of aggregation or definitive boundaries [START_REF] Pauliuk | Socioeconomic Metabolism as Paradigm for Studying the Biophysical Basis of Human Societies[END_REF]. Social ecology proposes representations at multiple scales, from local to global. In agroecology, scales such as the agroecosystem, the food system and the landscape are all considered as legitimate for investigation [START_REF] Francis | Agroecology: The Ecology of Food Systems[END_REF]. Some schools take a step further and consider that multiple representations should be taken into account at the same time: MuSIASEM explicitly considers the existence of different "value systems" found among stakeholders. Their incommensurability implies that multiple and irreducible representations are inevitable (Giampietro et This implies a shift from scientific-knowledge-based action to multi-criteria-debate-based one. [START_REF] Görg | Challenges for Social-Ecological Transformations: Contributions from Social and Political Ecology[END_REF] propose anchoring social ecology in critical thinking and transdisciplinarity with the help of political ecology. This leads to considering several goals together, e.g, composing industrial ecology's goals with animal health, pollution or diversity, thanks to a multi-criteria analysis [START_REF] Bonaudo | Agroecological Principles for the Redesign of Integrated Crop -Livestock Systems[END_REF]. [START_REF] Wassenaar | Reconsidering Industrial Metabolism: From Analogy to Denoting Actuality[END_REF], speaking about industrial ecology argues that goals or "desirable changes" "would best be based on locally debated criteria". Multi-faceted and composite representations adopt this posture more extensively:

MuSIASEM acknowledges that decisions have to emerge from negotiation and are not a prerequisite engraved in stone. Industrial ecology's goal of "closing the loop" is nuanced with other social goals (Boons et al. 2009). Pragmatic sociology, by taking an interest in the multiple representations of the actors and the particular situations in which they are linked, contributes to giving them a place in the social debate without imposing any normative goal.

Agroecology gives a central place to the knowledge and choices of farmers, and re-anchors the choices regarding food systems on a societal scale.

Conclusion

This work offers some cross-sectional analysis that may be of value for SEM practitioners with an interest in social changes, including sustainability or transdisciplinary practices. It provides an understanding of the multiple schools of thought and can be used to acquire a better grasp of their sometimes implicit presuppositions. It also allows them to be discussed in relation to their choices of representation of metabolism, and in terms of goals and means of action. Our results suggest an opportunity to move away from a positivist and valuefree epistemology in the study of SEM. Pragmatism provides tools to deal with the multiple incompatible values at play in the transformation of SEM, which echoes a vision of Ecological Economics with weak comparability of values (Martinez-Alier et al. 1998).

Acknowledgement

The PhD scholarship of A.W. Gabriel is funded by INRAE. This study received funding from the French Environment & Energy Agency (ADEME) through the BOAT project. We would like to thank the anonymous reviewers who contributed greatly to enhancing the quality of the article.

  al. 2018), a cross-sectional analysis between industrial ecology and politics (Breetz 2017), and tool oriented ones (Fernandez-Mena et al. 2016; Beloin-Saint-Pierre et al. 2017; Haberl et al. 2019).

2. 2 . 1

 21 The representation of socio-economic metabolism: stocks/flows/funds/scales levels and socio-economic context Articles are classified according to their SEM representation, especially the way they deal with (a) stocks, flows and funds; (b) scales and levels; and (c) socio-economic context. These three criteria are used to distinguish schools of thought.

Figure 1 :

 1 Figure 1: Scheme of research-management interfaces (Gosselin et al. 2018).

3

  Results: eight schools of thought to represent SEM and their links with actionWe identified eight schools of thought related to three different types of SEM representations.

  Industrial ecology stems fromAyres's (2002) analogy between the ecosystems of ecological sciences and the industry. SEM representations represent products or substance flows, and focus on economic agents at different scales: an individual agent, e.g., a collective facility[START_REF] Chance | The Plant-An Experiment in Urban Food Sustainability[END_REF]), a couple, e.g., an olive farm and a mill[START_REF] Zabaniotou | Boosting Circular Economy and Closing the Loop in Agriculture: Case Study of a Small-Scale Pyrolysis-Biochar Based System Integrated in an Olive Farm in Symbiosis with an Olive Mill[END_REF], or a large network of farmers or industries[START_REF] Nowak | Nutrient Recycling in Organic Farming Is Related to Diversity in Farm Types at the Local Level[END_REF][START_REF] Florin | Circular Economy in Romania: An Industrial Synergy in the Agri-Food Sector[END_REF]. Studies are divided between retrospective case studies (Nuhoff-Isakhanyan et al. 2017) and scenarios of industrial symbiosis (a real, linear, unsustainable system is compared to a closed and ideal system). For example, Alfaro et al. (2014) studied a small farming system at a village scale. A scenario of rural symbiosis was presented in the aim of increasing productivity and decreasing waste. While most studies do not rely on deep socio-economic analysis, some explore factors that are internal to economic agents such as knowledge, attitudes or practices.Multiple stakeholders are taken into account (e.g., farmers, extension agents)(Bellarby et al.

  . It recognizes that each description of the metabolism is necessarily partial. There are mutually multiple descriptions of the metabolism, provided by different scientific communities, mutually irreducible to each other and nevertheless relevant. Post normal science includes practices typical of research-within-management interfaces such as communication of uncertainties, taking part in social negotiation about desirable changes, and the co-evaluation of results within an extended peer community, including stakeholders. For example, Bruonori et al. (2016) assess local and global food chains across different commodities and countries through multi-scale metabolic and participatory evaluation. The results are then discussed in workshops with stakeholders.

Farmers

  are integrated into the construction of knowledge in participative action-research within a research-within-management interface (Méndez et al. 2013; Guzmán et al. 2012). For example, Moraine et al. (2017) propose a framework to perform integrated assessment of crop-livestock systems at the territorial level, combining ecological (crops, grasslands and animals) and social (farmers and chain actor interactions) systems. This framework is used as an intermediary object with stakeholders in participatory design approaches. However, many studies only provide elements of diagnosis or analysis in a trickle-down way. Resque et al. (2019) analyze agrobiodiversity, and how it relates to public mediated food chains.

  or convention theory (Kristensen et al. 2016; Coq-Huelva et al. 2012).

  ). They aim to bring out different visions of metabolism concerning its sustainability (Onyas et al. 2018), resilience (Wegerif et al. 2016) or possible future arrangements (Kristensen et al. 2016). These works are considered as performative by the researchers. Following the internal logic of the school, we consider these works as relying on research-within-management.

Figure 2 :

 2 Figure 2: Summary diagram of schools of thought according to modes of representation and action

  The different schools also influence each other. Social ecologists turn to multi-level analysis or downscale to get closer to actors[START_REF] Fischer-Kowalski | Conceptualizing, Observing, and Influencing Social-Ecological Transitions[END_REF]). Urban ecologists look for quantitative tools in industrial ecology (Beloin-Saint-Pierre et al. 2017). Social agrarian metabolism is described as a fusion between agroecology and social ecology, analyzing agroecosystem components through a metabolic lens, as funds and flows (González de Molina et al. 2020). Concepts such as agro-industrial ecology are proposed as mixes between agroecology and industrial ecology in the aim of more sustainable agricultural systems (Dumont et al. 2013; Fernandez-Mena et al. 2016).

  Wassenaar 2015; Pauliuk et al. 2015; Breetz 2017; Beloin-Saint-Pierre et al.2017). We suggest that an appealing path lies in the pragmatic philosophy.

4. 4 . 1

 41 Pragmatism, a philosophy anchored in empiricism This current of thought is born in the United States at the beginning of the 20th century with Charles Peirce, William James and John Dewey. It has made a major contribution in linking ideas and their consequences, and particularly their usefulness for action. The first pragmatists developed their ideas in opposition to the idealistic philosophers of their time. They criticized them for giving value to abstract concepts with no direct link to reality. To the idealistic attitude, pragmatism opposes an empirical one. The philosopher's work consists in inquiry, investigation. Ideas, concepts or representations have no value in themselves and must be tested in accordance with the facts. Thus, pragmatism is a generalization of the scientific and experimental spirit to the whole of our human experience. Pragmatists propose to focus on the practical, factual consequences of our ideas. They enjoin us to "consider the practical effects of the objects of [our] conception. Then, [to consider that our] conception of those effects is the whole of [our] conception of the object" (Peirce et al. 1923).

  al. 2009). The same position is defended by pragmatic sociology or urban and territorial ecology which describes agents with different representations of the reality (Buclet 2015; Latour et al. 2017). 4.4.3.4 Collective action and deliberation: multi-criteria-debate-based goals Concerning action, most schools value collective action and deliberation, to a certain extent.

  Altieri 2002; Bonaudo et al. 2014; Dumont et al. 2013; Francis et al. 2003; González de Molina et al. 2020; González de Molina et al. 2017; Guzmán et al. 2012; L. Resque et al. 2019; Martin et al. 2016; Méndez et al. 2013; Méndez et al. 2017; Moraine et al. 2017; Rivera-Ferre 2018; Rosset et al. 1997; Mier y Terán Giménez Cacho et al. 2018; Ryschawy et al. 2017; Vaarst et al. 2018; Wezel et al. 2018"

						Kowalski et al. 1998; Frankova 2017; Grešlová
						et al. 2019; Grešlová et al. 2015; Gueldner et
						al. 2017; Guzman et al. 2015; Guzman et al.
						2018; Kohlheb et al. 2009; Krausmann 2009;
						Krausmann 2004; Kuskova 2013; Magalhães et
						al. 2019; Soto et al. 2016; Yuan et al. 2011;
						Zhang et al. 2012"
	Metabolic rift and	Stocks and flows at	Change the system through trickle-	"Clausen et al. 2015; Foster et al. 2014;
	Marxist ecology (9)	large-scale spaces ana-lyzed through Marxist	down and critical analysis directed at civil society	Gomiero 2017; Gunderson 2011; Martinez-Alier et al. 2010; Moore 2000; Moore 2011; Schneider et al. 2010; Schneider 2017"
		frameworks
	Urban and territo-	Stocks and flows at mid-	Develop and relocalize the terri-
	rial ecology (10)	dle or small scales ana-	tory's economy through transfer-
		lyzed through stakeholder	and-translate and provision of man-
		analysis	agement tools directed at local au-
			thorities
	Supply-chains-
	based metabolism
	(11)
	Industrial ecology	Stocks and flows at in-	Close the loop and foster economic
	and symbiosis (15)	dividual companies or lo-	performance through user-push or
		cal scale analyzed through	transfer-and-translate and provision
		stakeholder analysis	of management tools directed at
			companies
	Multi-scale analysis	Economic and ecological	Preserve the funds through a
	of agroecosystems	funds and flows at mul-	research-within-management
	(8)	tiple scales analyzed by their social performance	interface and post-normal-science-based tools directed at various
			stakeholders
	Agroecology within	Ecological funds and flows	Develop	an	ecological-process-
	food systems and	at the food system and	based and socially fair food
	landscapes (18)	landscape scales analyzed through power structure	system through research-within-management interfaces directed at
		frameworks	farmers and civil society
	Pragmatic sociol-	Humans and non-humans	Enhance diverse and locally-based	"Akrich et al. 2006; Callon 1984; Coq-Huelva
	ogy and earthbound ecology (11)	as funds and flows along non-scalar biomass flows analyzed jointly as actor-	sustainability programs through research-within-management and extended descriptions directed at	et al. 2012; Glover et al. 2018; Goodman 2001; Gray et al. 2013; Kristensen et al. 2016; Le Velly et al. 2016; Mol et al. 2006; Onyas et al. 2018; Wegerif et al. 2016"
		networks	human and non-human entities

"Bouwman et al. 2013; Cusso et al. 2006; Díaz de Astarloa et al. 2018; Fischer-Kowalski et al. 2009; Fischer-Kowalski et al. 2015; Fischer-"Barles 2014; Barles et al. 2011; Barles 2015; Barles 2007; Billen et al. 2012; Buclet 2011; Buclet 2015; Cerceau et al. 2014; Oliveira et al. 2016; Tedesco et al. 2017" Economic-agent-based representations (26) Stocks and flows at individual companies or sector level analyzed through stakeholder analysis Limit environmental impact and foster economic performance through user-push or transfer-and-translate and provision of management tools directed at companies and trade associations "Blengini et al. 2009; Courtonne et al. 2016; Filippini et al. 2016; Kulak et al. 2016; Kytzia et al. 2004; Miranda-Ackerman et al. 2017; Pagotto et al. 2016b; Sellitto et al. 2018; Wirsenius 2003; Xu et al. 2016; Yazan et al. 2018" "Alfaro et al. 2014; Bellarby et al. 2017; Multi-faceted and composite representations (37) "Brunori et al. 2016; Gamboa 2011; Giampietro 2004; Gomiero et al. 2001; González-Acevedo et al. 2016; Grillot et al. 2018; Scheidel et al. 2015; Serrano-Tovar et al. 2014" "

Table 2 :

 2 Articles classified in the eight schools of thought.

NB: The 7 criteria used are made explicit within the table

metabo* means that all words starting with metabo were investigated, e.g., metabolism, metabolic, metabolized, etc.

Chance et al. 2018; Fernandez-Mena et al. 2019; Frone et al. 2017; Hobbes et al. 2007; Iacondini et al. 2015; Niutanen et al. 2003; Nowak et al. 2015; Nuhoff-Isakhanyan et al. 2017; Pagotto et al. 2016a; Shastri et al. 2011; Simboli et al. 2015; Tamura et al. 2014; Zabaniotou et al. 2015"


Colin R Tinsley
email: tinsley@grignon.inra.fr

Emmanuelle Bille

Xavier Nassif
email: nassif@necker.fr

Bacteriophages and pathogenicity: more than just providing a toxin

Keywords: Neisseria meningitidis, bacteriophage, genetics, evolution, pathogenesis

An increasing number of pathogenicity factors are discovered to be carried by bacteriophages. This review considers the bacteriophage-bacterium interaction and its relation to disease processes. We discuss the search for new bacteriophage-associated pathogenicity factors with emphasis on recent advances brought by the use of genomic sequence data and the techniques of genomic epidemiology.

Introduction and historical perspective

Horizontally mobile elements, including plasmids, transposons, genetic islands, and bacteriophages are responsible for the transfer of new functions to a bacterial cell and are recognised as important agents in bacterial evolution. To be maintained as part of the genetic content of the bacterium the genetic unit must either encode a mechanism by which its inactivation kills the cell (eg, restriction-modification or post-segregational killing systems), or must provide a benefit to the organism. Typically this involves coding for resistance to antibiotics, detoxification of heavy metals, acquisition and utilisation of certain nutrients, evasion of predators or colonisation of specific environments. In the case of human disease, the most important factors are colonisation, acquisition of nutrients, evasion of the innate and adaptive immune response, and pathogenicity factors. This review will discuss the involvement of bacteriophages in the transfer of pathogenicity factors.

Numerous examples have been brought to light of modification of bacterial virulence by bacteriophages. The first demonstration was that of Freeman [1], who found that avirulent strains of Corynebacterium diphtheriae infected with bacteriophage (now known as corynephage beta; Fig. 1) yielded virulent lysogens that produced diphtheria toxin. Later work showed that the gene was carried by the bacteriophage, the expression of which was intimately linked with genetic regulation (the Fur homologue, DtxR) in the host bacterial cell. Zabriskie demonstrated that the filtrable agent described by Frobisher in 1927, inducing production of streptococcal pyrogenic exotoxin (scarlatina toxin) by non-toxinogenic streptococci, was a temperate bacteriophage [2]. A further example is furnished by Shiga toxin-producing (enterohaemorrhagic) Escherichia coli, where systemic circulation of toxin may lead to renal failure and haemolytic uraemic syndrome (HUS). In this case, toxinogenicity was shown to be transferable between strains by a bacteriophage vector [3]. More recently Waldor and colleagues, [4] demonstrated that the cholera toxin was encoded on a filamentous bacteriophage, CTX-phi.

The place of the bacteriophage in the biology of the microbe. Regulation of virulence and phage particle production.

The case of CTX-phi highlights many of the important themes in bacteriophage-carried pathogenesis factors. The major bacterial determinants of choleric disease are the pili (composed of subunits known as TCP = toxin-coregulated pilin) which permit colonisation of the human intestine, and the secreted toxin, CTX, which is responsible for the life-threatening diarrhoea. The pili are encoded as part of a pathogenicity island of about 40 kb, VPI [5] and, in addition to mediating adhesion to enterocytes, serve as receptors for infection by the bacteriophage CTX-phi. This bacteriophage, resembles phage Ff (Ff is the collective designation for the nearly identical coliphages f1, fd and M13) in size and genetic organisation, but has toxin genes in place of the bacteriophage secretin, co-opting part of the bacterial type II secretion system for its release from the bacterial cell. Unlike Ff, the bacteriophage is carried as a prophage integrated into the bacterial chromosome. The production of bacteriophage particles is dependent on a particular, tandem arrangement of bacteriophage elements [6], and is maintained at a low level by the repressor protein (reviewed by [7]). The virulence factors, TCP and CTX, are part of the Tox regulon, a twocomponent regulatory system integral to the bacteria which, in response to external signals, directly activates expression of the toxin genes and, via ToxT (encoded on VPI), activates both the toxin genes and the TCP pilus operon. Other VPI-encoded factors link the production of toxin and flagellar motility with quorum sensing. This example shows how bacteriophages specifying pathogenicity phenotypes can be integrated into the genetic regulatory circuits of the host bacterium. This fine-tuning of the expression of phenotypes carried by the lysogenic bacteriophage is important to minimise the deleterious effects of virus-carriage and to optimise the advantage (competitive or in terms of colonisation of new environments) imparted by the bacteriophage pathogenicity genes.

Many pathogenesis factors are carried on bacteriophages related to coliphage lambda or on other temperate bacteriophages that switch from a lysogenic state to a lytic cycle. The expression of bacteriophage-associated pathogenicity genes may again be controlled by promoter regions responsive to elements of the host's genetic regulatory systems but is also strongly affected by induction of the bacteriophage, which in this case leads to death of the bacterial host. Bacteriophage replication increases the copy number of bacteriophageassociated pathogenesis genes, and also causes cell lysis, which results in release of large quantities of intracellular toxin. Thus, in the case of Corynebacterium diphtheriae, toxin production is derepressed under low iron conditions, as mentioned above, and is greatly increased after induction of the bacteriophage [8]. Similarly the genes stx in Shiga toxinproducing (enterohaemorrhagic) Escherichia coli are situated among genes controlled by the late promoter and are therefore cotranscribed with the lysis genes of the bacteriophage. Shiga toxin 1 (Stx1) is also regulated by iron concentration, whereas Stx2 is not [9]. These bacteriophages are also responsive to elements of the bacterial SOS system, DNA damage, leading to cleavage of the bacteriophage repressor by RecA binding ssDNA (This is a common response of bacteriophages, including CTX-phi). These regulatory systems are relevant in the context of human disease. Pathogens are likely to encounter iron-limited conditions imposed by the human organism as a defence against invading bacteria. In addition DNA damage may result from contact with bactericidal molecules produced by immune cells. Indeed, Broudy and colleagues [10] have shown that the temperate bacteriophage carrying the streptococcal pyrogenic exotoxin C was induced during co-culture of Streptococcus pyogenes with human pharyngeal cells, resulting in the release of large quantities of the toxin. Similarly interaction of STEC with neutrophils or with hydrogen peroxide (a neutrophil bactericidal product) induced both production of bacteriophage particles and secretion of Shiga toxin [11]. Equally, induction of toxin production has been seen to result from the use of DNA-damaging antibiotics ( [12] and references therein), leading to questions about their use in cases of gastroenteritis.

Survival of the bacteriophage, survival of the host bacterium

Since induction of the SOS response implies a high probability of death of host bacterium or of genetic damage to the prophage, there is an advantage in attempting to produce phage particles. In this case the survival of the phage, the fate of the bacteria or the human host is of no relevance, though the disease symptoms, diarrhoea for example, may help to disseminate the bacteriophage, within or without the bacteria. On the other hand, accessory pathogenesis genes carried by the bacteriophage are usually transcriptionally autonomous, and any benefit that they may bring to the bacterium is at least partially independent of the host bacteriophage. Hence mutations which inactivate genes involved in bacteriophage replication but leave the pathogenesis (or immunity) genes intact will be advantageous for the bacterium. Indeed prophage remnants are commonly found in genome sequences, and for example the majority of the lambdoid prophages in STEC are non-inducible. Another, interesting example are the R-and F-type pyocins of Pseudomonas aeruginosa, which appear to have evolved from the tail fibres of bacteriophages [13], the target in this case being competing bacteria. Thus genes transferred horizontally by bacteriophages, if they provide an advantage to the lysogen in a particular environment will tend over time to become dissociated from the bacteriophage functions and will end by being "appropriated" as part of the bacterial core chromosome, as in the model of the role of "pathogenicity islands" in the evolution of pathogens [14]. In keeping with this notion, the stx genes in certain strains of Shigella are encoded within a degraded prophage [15]. Striking exceptions to this are the bacteriophages of Streptotoccus pyogenes, which carry a variety of pathogenicity factors, for example the superantigen streptococcal pyrogenic exotoxin. In one serotype M3 strain, all pathogenicity factor-encoding prophages were inducible [16], suggesting a strong selection for mobility of these elements.

Whether a virus kills the bacterium by lysis, or is secreted without causing cell death, a time will come when the local population of bacteria is either killed or resistant. The bacteriophage particles, generally very resistant in the environment, may encounter a new susceptible population, but lysogeny provides a second means to promote survival. The propensity of the bacteria for transmission to a new human habitat will not only lead to an increased number of prophage genomes as the bacteria colonise the new host, but will also bring the bacteriophage into contact with new, potentially susceptible populations of bacteria. During the time spent integrated in the bacterial chromosome however, the bacteriophage is dependent on the host cell and carriage of the bacteriophage will add a metabolic burden to these bacteria and/or kill a proportion by lysis, potentially rendering the bacteriophagebacterium association less competitive. This effect will be counteracted if the bacteriophage brings some form of selective advantage to the lysogenised bacterium. Thus, the inherent advantages of being a lysogen are immunity to lysis by infection with the same or similar bacteriophages and killing of competing bacteria by phage particles released from a small fraction of the lysogens. A further advantage may be achieved when the bacteriophage carries additional genes of benefit to the bacterium.

Functions carried by bacteriophages

Analysis of the first studied of the lambdoid bacteriophages, lambda itself, has indeed identified such beneficial genes among the "accessory" sequences, dispensable for phage growth and comprising a sizeable proportion of the 48.5 kilobase genome. The Bor and Lom gene products, homologous to other chromosomally coded bacterial pathogenicity factors, are involved respectively in adhesion and in resistance to the host immune system. Another pair of genes rexA and rexB provides some immunity to a variety of unrelated bacteriophages. Hence the "original" lambda encodes factors which promote survival of the lysogen. Comparative virus genomics has uncovered many other such accessory genes, present in a subset of related viruses [17]. These include genes for the Shiga toxin, superoxide dismutase and SopE, an effector injected by the type III secretion system of Salmonella.

The list of pathogenesis determinants carried by bacteriophages continues to grow. Various surface proteins promoting adhesion are virus encoded. Bacteriophage-associated invasins include, as mentioned above, effectors important in uptake into mucosal cells, and also enzymes which cause degradation of the extracellular matrix to allow dissemination of the bacteria from the focus of infection in invasive streptococcal and staphylococcal disease. A group of bacteriophages in the enterobacteria code for enzymes which, interacting with the host cell biosynthetic machinery, alter the antigenic structure of the O side chain of the LPS, hence modifying interactions with human cells and providing a mechanism of avoidance of the immune response. Certainly the most well-known of the bacteriophage-carried pathogenicity factors are the exotoxins: diphtheria, Shiga and cholera toxins, certain of the botulinum toxins (predominantly associated with avian botulism rather than with foodborne intoxications) and a plethora of staphylococcal and streptococcal toxins involved in invasive disease. Indeed the importance of bacteriophages in the evolution of bacterial virulence is exemplified by the case of Streptococcus pyogenes, where an accumulation and reassortment of virus-encoded pathogenicity factors is probably responsible for the emergence of particularly virulent clones causing highly invasive disease [18].

An interesting alternative means by which bacteriophages may contribute to virulence is afforded by the recent example of the involvement of Pseudomonas aeruginosa filamentous bacteriophage Pf4 in the formation of biofilms [19]. Here, localised activation of the prophage (or spontaneous mutation to resistance to the bacteriophage repressor system) causes a decrease in growth rate in affected cells, and also to regions of cell death in the biofilm. The regions of cell death correspond to the voids characteristic of the three-dimensional structure of pseudomonal (and certain other) biofilms. It is tempting to speculate that this behaviour is adaptative in the context of the biofilm, participating in the genesis of the open structure increasing nutrient transport, and allowing dissemination of colonising bacteria (These regions of cell death nevertheless contain viable cells which are released from the biofilm). It is known that P. aeruginosa grows as a biofilm in the lungs of persons suffering from cystic fibrosis, and so it is possible that this filamentous phage plays a role in the pathogenesis. For further discussion of bacteriophage-carried pathogenicity factors, the reader may consult a number of recent reviews, for example [9,20,21].

Discovery of new bacteriophage-carried pathogenesis determinants

The discovery of the association of these pathogenicity factors with bacteriophages has involved different methods, depending on the historical context. For example, the discovery that the diphtheria toxin was bacteriophage-encoded proceeded, from the discovery of Freeman and Morse that toxin production was due to lysogeny by bacteriophage beta, through the production of mutants, genetic and physical maps of the bacteriophage [22], to sequencing of the tox gene [23] which opened up the possibility of the engineering of immunotoxins, for example. In an inverse procedure, based on the observation of tandem arrays of CTX operons in V. cholerae [6,24], Waldor and Mekalanos discovered that the toxin genes were part of a filamentous bacteriophage, and that the capacity to produce cholera toxin was transferable between strains. Advances in sequencing techniques and in particular the advent of genome sequencing allow the relatively facile identification of prophage sequences in genomes.

Nevertheless, identification is complicated by the fact that some conjugative transposon-like elements contain bacteriophage-like integrases, as do integrons and pathogenicity islands. Again, nucleotide sequence homologies may more or less strongly suggest that bacteriophage genes are involved in pathogenesis, but the large number of open reading frames (ORF) of undetermined function in bacteriophages [17] leaves room for speculation concerning the possible involvement in pathogenesis of ORFs lacking homology to proteins of known function.

Pathogenicity factors carried by bacteriophages are then a subset of genetically mobile pathogenicity factors and new, phage-encoded pathogenicity factors are amenable to detection by standard techniques. These include random transposon mutagenesis, in vitro expression technology, and systematic gene inactivation, using tissue culture or animal models of pathogenesis. In a recent example, Gonzalez and colleagues used signature-tagged mutagenesis to identify factors necessary for pathogenesis of Escherichia coli serotype K1 in the infant rat. Investigation of one of the pathogenicity factors so identified showed that it was part of a region with similarities to filamentous bacteriophages [25], and indeed bacteriophage particles were found to be secreted into the supernatant. By a search of available genomic sequences, a similar sequence was found in the clone orientalis of Yersinia pestis (enterobacteriaceae) responsible for the current plague pandemic.

Thus, biological assays screening banks of mutants produced, for example, by random transposon mutagenesis, signature-tagged mutagenesis provide powerful methods of detection of pathogenesis-associated genes. However, production and screening of the bank is very labour-intensive. This is a particular problem for in cases where there is no animal model that adequately represents the disease as it occurs in humans, and where a search for pathogenicity factors therefore necessitates screening in several assays representing different stages of the disease process. The advent of the "genomic era" provides a wealth of information permitting the choice of candidate genes, for example on the basis of similarity to known pathogenesis factors. On the other hand, the large quantity of information demands that methods be developed to find the most relevant candidates. Apart from the possible errors of attribution of function based on frequently partial homologies, many predicted genes are without homology to genes of known function. The choice of potential pathogenicity factors therefore remains a considerable task. Comparative genomics provides a method of narrowing down the range of possible candidates by choosing genes specifically present in a population expressing a particular pathogenicity phenotype. Whole genome DNA arrays allow the detection of the presence or absence of each gene in the genome in a range of isolates of varying pathogenic potential. This method will not detect relatively small genetic differences, but will reveal the presence or absence of all but the smallest genes and will certainly bring to light pathogenicity islands and bacteriophages.

Molecular epidemiological solutions: the case of Neisseria meningitidis

Such approaches have been used to search for new pathogenicity factors associated with certain disease phenotypes. A comparison of strains of Vibrio cholerae associated with epidemic versus non-epidemic disease brought to light several groups of genes including a type III secretion system [26]. Analysis of isolates of Helicobacter pylori associated with different outcomes of infection [27] showed great variability in gene content, and the authors were able to identify several putative pathogenesis determinants. Neisseria meningitidis is another bacterial species with differences in pathogenic potential between individual clones. Colonisation by the meningococcus usually leads to an asymptomatic infection of the nasopharyngeal mucosa, but in a small proportion of cases the bacteria cross the epithelium, gain access to the bloodstream and disseminate to cause systemic disease which is typically characterised by a cerebrospinal meningitis. The reasons for which some meningococcal strains cause life-threatening disease remain unclear. Disease is a multifactorial process, depending on characteristics of both the host and the infecting bacteria. Several pathogenicity factors are well known, for example the adhesive pili which permit the attachment to the nasopharyngeal epithelium, the polysaccharide capsule which affords protection against the immune system and allows survival in the blood, and the iron-acquisition systems based on the transferrin-and lactoferrin-binding proteins. However these factors are widely distributed among meningococci irrespective of their pathogenicity and can not by themselves explain the differences in pathogenic potential between isolates of this species. It is likely that the capacity to cause disease is determined by the combination of a number of pathogenicity factors, and that some surely remain to be discovered. Comparison of strains of different pathogenic potential by the use of whole genome DNA arrays is an attractive strategy to accomplish this. The use of molecular genetic techniques such as Multi-Locus Sequence Typing [28] confirmed earlier observations that disease-causing isolates were restricted to a small number of the many clonal groups -the so-called "hyperinvasive" lineages -and hence permits the choice of isolates corresponding to meningococci of defined pathogenic potential for use in DNA array experiments.

N. meningitidis is a naturally-transformable species; genetic change is predominantly due to horizontal transfer between different strains, and the population structure of the meningococcus is web-like rather than tree-like (Fig. 2). Nevertheless successful clones arise and spread within the human population, where they survive for a matter of years before herd immunity leads to their being replaced by new strains. Some of these clones have relatively high pathogenic potential (measured as attack rate, or case/carrier ratio) and will give rise to epidemics of meningococcal disease. Others are probably non-pathogenic, while the majority of groups comprise bacteria of different degrees of pathogenicity, or too few isolates have been collected to allow an estimation of their potential to cause disease. Hence we can compare the genetic complement of isolates from several of the so called "hyperinvasive" clonal groups with that of isolates from clonal groups that have been associated only with asymptomatic carriage, which we may call "non-invasive" groups (presuming them to be incapable of causing disease). Indeed a whole genome DNA array comparison of 48 strains belonging to clonal groups of high pathogenicity with 19 strains belonging to clonal groups never having caused disease [29] brought to light a genetic island of about 8 kb that was specifically present in the isolates belonging to pathogenic clonal groups (Fig. 3). Blast comparisons against the public databases revealed similarity of the first ORF with the phage replication proteins of phages CTX and Ff. This, combined with a similar genetic organisation suggested that the genetic island corresponds to an integrated bacteriophage genome. Figure 3 summarises the similarities between the meningococcal prophage, the archetypal coliphage Ff and other filamentous bacteriophages recently associated with pathogenicity.

The presence of the prophage in a number of unrelated hyperinvasive lineages and its absence from a number of non-invasive lineages suggests a causal relationship (the probability that the results were due to chance alone was estimated to be 0.7%). However, the demonstrated association is with the hyperinvasive epidemiologial groups rather than with the disease itself. In addition, if the prophage were associated with another, hypothetical, gene that increased the pathogenic potential of the meningococcus, a large proportion of bacteria belonging to the same clonal complex would contain both the prophage and the pathogenicity gene and would have a higher attack rate independent of the effect of the prophage. However, N. meningitidis being a naturally transformable bacterium, gene transfer will occur horizontally as well as vertically (clonally). Furthermore a bacteriophage, being an infectious DNA molecule has evolved to pass horizontally between individual organisms. If the prophage contributes to pathogenicity, bacteria carrying it will tend to cause disease more frequently than do bacteria without the element, though the effect may not be striking (since the most virulent organisms cause disease in only a small proportion of those people who are colonised).

The DNA array analysis was therefore followed by a larger epidemiological analysis of 193 strains taken from a study of carried strains in the Czech Republic [30]. The isolates were classified according to three characteristics: having or not having caused disease, possessing or not possessing the prophage (defined by PCR and/or Southern blot analysis) and belonging to or not belonging to a recognised hyperinvasive clonal complex. As discussed above, each of these variables affects the other two, but multivariate analysis permits an estimation of the strength of association of each of the pairs in the absence of an effect from the third. Hence the effect of the presence of the prophage on the attack rate of the meningococci is highly significant (p < 0.001) even taking into account the effect of the clonal relationships within the population studied. Meningococci carrying the prophage are more likely to cause disease.

Hence the prophage is apparently a pathogenicity factor for Neisseria meningitidis. However, once the prophage had been identified, similarity searches demonstrated little homology to known virulence factors (ORF1 was homologous to the CTX-phi phage replication protein, ORF8 showed some similarity to the phage assembly protein Zot and ORF9 is related to transposases). This contrasts with the case of bacteriophage CTX-phi, which carries a defined toxin, and that of the E. coli / Yersinia bacteriophage, which was identified after sequencing the flanking region of puvA, whose inactivation led to reduced virulence in an animal model. The question remains then as to the advantage brought by the prophage to the host cell, and how this causes the bacteria to be more pathogenic. By analogy with CTX-phi, or the E. coli (and Y. pestis) bacteriophage, the genes ORF6 and ORF9 of the meningococcal prophage would be the most attractive candidates (see Fig. 3), but in the absence of experimental evidence any of the genes might have an effect on the interaction of the bacteria with the host. The membrane proteins will come into contact with the host cells and immune effectors, while it is possible that the transposase ORF9 serves to maintain a high level of genetic fluidity, which may be beneficial to the clonal group as a whole.

Experiments attempting to demonstrate a possible effect of the bacteriophage on meningococcal disease have not so far been conclusive. Indeed deletion of the entire prophage did not have any effect in laboratory models of meningococcal pathogenesis [29], suggesting that the effect of the bacteriophage is either subtle, or not detectable by the tests used. It is certain that the balance between commensalism and pathogenicity is delicate in the case of Neisseria meningitidis. A small increase in resistance to the human's immune defence system, induced by the bacteriophage, giving the meningococci a competitive edge in the habitat of the nasopharyngeal mucosa might tip the balance from a relationship of commensalism to one of a pathogen-host conflict. Fig. 1. Corynephage beta released from toxinogenic C. diptheriae are capable of transforming non-pathogenic strains to toxin-releasing strains which are virulent in animal models. Reprinted with permission from [1]. Fig. 2. Schematic phylogenies of (a) a predominantly clonal bacterium (eg. Escherichia coli) and (b) a bacterium undergoing frequent horisontal transfer (eg. the naturally-transformable Neisseria meningitidis) as would be produced by split decomposition analysis of multienzyme sequence data. Isolates of higher pathogenic potential (red) are readily identified in the clonal phylogeny as clades, related by descent from a common, pathogenic ancestor. The most economical interpretation is the acquisition of one or more pathogenicity factors (arrows) by the common ancestor. In the case of the transformable species the genetic information, including the phylogenetic markers, of one isolate may come from more than one "parent", complicating the interpretation of the phylogeny. However, clonal expansion of certain genotypes will nevertheless lead to distinguishable groups of related bacteria (for example, at the top of the figure).

Fig. 3 .

 3 Fig.3. Organisation of genes in the prophage form of filamentous bacteriophages which have been associated with pathogenesis and comparison with coliphage Ff. Homologous genes (having predicted protein products showing similarity by BlastP) are similarly coloured. Striped and stippled colouring indicates low homology. Green are phage replication proteins, orange are repressors, gene I/XI of coliphage Ff (violet) is involved in the export of bacteriophage particles. ORFs in N. meningitidis, Y. pestis and P. aeruginosa are as predicted by the genome sequencing projects; those of V. cholerae are modified with reference to[7]. The sequence of the prophage from E. coli was not available.

  Ff (violet) is involved in the export of bacteriophage particles. ORFs in N. meningitidis, Y. pestis and P. aeruginosa are as predicted by the genome sequencing projects; those of V. cholerae are modified with reference to[7]. The sequence of the prophage from E. coli was not available.

	N. meningitidis N. meningitidis	NMA1792 NMA1792	93 94 95 96 93 94 95 96	97 97	98 98	99 99	1800 1800
	V. cholerae V. cholerae	VC1464 1463 VC1464 1463	1462 1462		1461 1461	1460 1460	1459 1459	1458 1458	1457, 1456 1457, 1456
		rstR	rstA	rstB	psh	cep	gIII	ace	zot	ctxAB
	Y. pestis	YP02271	72	73	74	75 76	76a	77	78	79	2280
									(puvA)
	E. coli K1 E. coli K1
									puvA puvA
	P. aeruginosa PA0715 P. aeruginosa PA0715 P. aeruginosa PA0715	16 16 16			17 18 17 18 17 18	19 19 19	20 21 22 23 20 21 22 23 20 21 22 23	24 24 24	25 25 25	26 26 26	27 27 27	28 28 28	29 29 29
	phage Ff phage Ff phage Ff			II/X II/X II/X	V V V	VII VII VII	IX VIII IX VIII IX VIII	III III III	VI VI VI	I/XI I/XI I/XI	IV IV IV
	(circular) (circular) (circular)
		1 1 1	2 2 2		3 3 3	4 4 4		5 5 5	6 6 6	7 7 7	8 8 8	9 9 9	10 Kb 10 Kb 10 Kb

Acknowledgements

We thank Dr. Serge Cassaregola for careful reading of the manuscripts and helpful comments.