This special issue of Biofilms highlights current experimental studies and modeling of collective phenomena in single-celled microbes, primarily bacteria, stemming from the workshop Biocomplexity VI: Complex Behavior in Unicellular Organisms, held May 12th – 16th, 2004 at Indiana University, Bloomington, and co-organized by the Biocomplexity Institute at Indiana University and the Interdisciplinary Center for the Study of Biocomplexity at the University of Notre Dame, with support from the National Science Foundation, the National Institute of General Medical Sciences and the College of Arts and Sciences at Indiana University.

The collective behavior of bacteria is significant in bioengineering, agriculture, medicine, dentistry and geosciences, among other disciplines. Microbes exhibit complex behavior through motility, cellular differentiation and host interactions (both symbiotic and pathogenic). Microbial biofilms are one of the major products of complex microbial behaviors. Biofilms can cause biofouling (which can be a major problem for both surgically implanted stents and submarines) and antibiotic resistance. Underlying these complex behaviors are intricate intercellular and intracellular signaling pathways. These pathways differ significantly from those in eukaryotes and thus present an important opportunity to compare different natural design solutions to similar biological needs. Thus the study of emergent behaviors such as biofilm formation and cellular differentiation in simple unicellular organisms can provide insights into both the evolution and function of multicellular patterning and development.

The Workshop discussed current and future problems in collective phenomena in single-celled organisms: quorum sensing and intercellular signaling, cell motility and taxis, microbial differentiation, pattern formation in simple eukaryotes, multicellular aggregation, distributive behavior, pathogenesis and symbiosis, and the evolution of bacterial cooperativity, including biofilms. The talks covered length scales from single molecule interactions and genetics to systems biology and ecology. A specific goal of the workshop was to bring together experimentalists and modelers, engineers and basic researchers who would not normally attend the same conference. This disciplinary mosaic aimed to promote cross fertilization to improve our understanding of the fundamental biology of collective behaviors, to lead to more useful models and engineering techniques, to initiate new modeling efforts, to promote collaborations between experimentalists and modelers, to transfer best practice between subdisciplines and to encourage more holistic approaches to problem solving. This combination of approaches seemed particularly appropriate because modeling of complex microbial behaviors such as biofilm formation is one of the more advanced and successful examples of multiscale biological simulations. Understanding the extreme complexity of microbial interactions for industrial, medical and agricultural applications requires a solid conceptual and computational framework.

This special issue of Biofilms contains fourteen papers from participants in the Biocomplexity VI Workshop. These papers illustrate the breadth and depth of studies presented at the Workshop. Three papers review important areas in unicellular complexity. Picioreanu, Xavier, and van Loosdrecht review recent advances in the mathematical modeling of bacterial biofilm formation. This group has pioneered many of the modeling advances in this area, and their review specifically highlights modeling approaches for multispecies biofilms. Ben-Jacob, Matsushita, and their colleagues, contribute separate reviews on patterned bacterial colony formation. Ben-Jacob, Aharonov and Shapira discuss emergent properties of bacterial colonies, while Matsushita integrates empirical data with current models of colony expansion.

Four additional contributions in this journal issue present new results from modeling and simulations of biofilm formation and cellular aggregation. Laspidou and Rittman describe a new modeling approach applied to monospecies biofilms that includes not only bacterial cells but the active and inert biomass they produce. Xavier, again with Picioreanu and van Loosdrecht, presents simulations using agent-based models to generate different biofilm morphologies under variable environmental conditions. Takhistov and George use empirical data from Listeria monocytogenes to model the early stages of microbe-surface and microbe-microbe interactions and their impact on the eventual structure of biofilms. Wimpenny and Colasanti, apply a cellular-automaton approach to aggregation between different bacterial species.

Five of the contributions in this issue present new experimental results on unicellular complexity. Papers from Kreth, Lux, van de Mortel and their colleagues employ technical advances to elucidate new features of microbial biofilms. Using a novel biofilm cultivation format, van de Mortel, Chang and Halverson examine the impact of desiccation, measured as matric stress, on cells within a Pseudomonas putida biofilm. Coupling confocal microscopy with vital staining, Lux and colleagues examine the spatial distribution of cells within the fruiting bodies of Myxococcus xanthus and observe a scaffold of exopolysaccharide, perhaps, supporting the three-dimensional fruiting body. Kreth and colleagues apply a battery of techniques to examine biofilm formation by the dental pathogen Streptococcus mutans, using a quartz crystal microbalance to measure surface-adhered biomass and microjet impingement to gauge the adhesiveness of the structure. Fontana, Haider and González-Cabezas also study S. mutans biofilms, using standard microbiological approaches, as they form on enamel in combination with four other oral microbes and find stable communities including all five species. Finally, Ohnishi and Horinouchi analyze the genetics of the complex A-factor signaling cascade that controls antibiotic synthesis and other forms of secondary metabolism in Streptomyces griseus.

The outstanding scientific range of the Biocomplexity VI workshop helped identify a number of important areas for future research. Although the connections between mathematical modeling efforts and experimental science are becoming more intimate, a significant challenge remains in relating real, complex biofilms to experiments and simulations that attempt to extract their salient features. What are the key parameters that dictate the ultimate configuration and activity of microbial populations and how do these parameters change over time? Microbes seldom exist in homogeneous populations; how do the behaviors of monospecies laboratory experiments and modeling relate to the greater complexity of multiple species interactions? Some of the papers in this issue address behavior in multispecies assemblies; future studies will continue this trend. Palmer, Diaz and Kolenbrander propose that dental biofilms, with their 600+ interacting species may be ideal for a top-down, systems-oriented approach, which integrates modeling and experiment.

Finally, few studies have applied evolutionary theory to microbial behavior. The concepts of individual reproductive success, competition and cooperativity are essential to studies of metazoan systems. What are the evolutionary selective costs and benefits of microbial behaviors? Kreft discusses the differences between group-selection and individual-selection mechanisms in biofilms and identifies some common errors in simplistic evolutionary explanations of bacterial behaviors. An evolutionary context is critical for us to move from describing the complex behaviors of microbes, to understanding why this complexity has evolved.

Clay Fuqua
James A. Glazier
Yves Brun
Mark S. Alber