Research Articles
Introduction to Proceedings of the Workshop “Biocomplexity VI: Complex Behavior in Unicellular Organisms”
- C. Fuqua, J. A. Glazier, Y. Brun, M. S. Alber
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- Published online by Cambridge University Press:
- 05 April 2005, pp. 227-228
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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 12–16 May 2004, at Indiana University, Bloomington, IN, USA, and co-organized by the Biocomplexity Institute at Indiana University and the Interdisciplinary Center for the Study of Biocomplexity at the University of Nôtre Dame, with support from the National Science Foundation (grant no. 0352904), the National Institutes of Health (grant no. GM071709-01), the National Institute of General Medical Sciences and the College of Arts and Sciences at Indiana University.
Caries lesion development and biofilm composition responses to varying demineralization times and sucrose exposures
- M. Fontana, A. Haider, C. González-Cabezas
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- Published online by Cambridge University Press:
- 26 January 2005, pp. 229-237
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The aim of this research was to study the effect of varying incubation times and sucrose exposures on lesion development and biofilm composition using a multi-species biofilm caries model. Two studies were conducted. In study 1, enamel specimens were divided into four groups, inoculated with a mixed overnight culture of Streptococcus mutans, Lactobacillus casei, Actinomycesnaeslundii, Streptococcusparasanguis and Streptococcussalivarius, and exposed to circulating trypticase soy broth + 5% (w/v) sucrose (TSBS; 30 min, three times per day (3 × /day)) and a mineral washing solution containing 0.25 p.p.m. fluoride (22.5 h/day) for 2, 5, 6 or 8 days. In study 2, additional enamel specimens were divided into four groups and exposed to the same biofilm for 7 days, but with variations in the feeding schedule: TSBS 3×/day for 5 min, TSBS 3×/day for 15 min, TSBS 3 × /day for 30 min, or TSBS 3×/day for 30 min + 1×/day for 15 min. At the end of each study, bacterial colonization counts and lesion size were determined. In study 1, specimens developed significantly deeper carious lesions with longer demineralization time (average lesion depth was 52.16 μm, 67.86 μm, 84.91 μm, and 99.97 μm, respectively, for 2, 5, 6, and 8 days). In study 2, there was no significant difference in size between the lesions developed at feeding schedules of 3 × /day for 5 or 15 min. Lesions exposed to longer (30 min) and more frequent feeding schedules (4×/day) were significantly larger than the other groups. In both studies, all five bacterial species were able to colonize the enamel and were present in all groups at the end of the experiments, with predominance of lactobacilli over S. mutans. In conclusion, larger lesions were observed with increased incubation time and more frequent feeding schedules, with small variations in biofilm composition.
Review Article
Bacteria harnessing complexity
- E. Ben Jacob, Y. Aharonov, Y. Shapira
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- 07 March 2005, pp. 239-263
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The study of bacterial colonies is a crucial step towards understanding biofilms. We review some of the exciting discoveries about bacterial self-organization that might shed new light on biocomplexity in general and biofilms in particular. This review is aimed at researchers from different disciplines – microbiology, biology, chemistry, physics, mathematics and computer science. To make the presentation comprehensible we have avoided the use of specialized terminology of the different disciplines and limited the experimental and computational details.
Bacteria can self-organize into hierarchically structured colonies of 109 to 1012 bacteria, each utilizing a great variety of biochemical communication agents, such as simple molecules, polymers, peptides, complex proteins, genetic material and also “cassettes of genetic information” such as plasmids and viruses. Bacteria use their intracellular flexibility, involving signal transduction networks and genomic plasticity, to collectively maintain self and shared interpretations of chemical cues, exchange of meaning-bearing chemical messages, and dialogues. The meaning-based communication permits the formation of colonial intentional behavior, purposeful alteration of colony structure and decision-making – features we might begin to associate with bacterial social intelligence. Such social intelligence, should it exist, would require going beyond communication to encompass additional intracellular processes, as yet unknown, for generating inheritable colonial memory and commonly shared genomic context.
Conflicts of interest in biofilms
- J.-U. Kreft
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- 25 January 2005, pp. 265-276
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High cell density and close proximity of diverse species of microorganisms are typical of life in natural biofilms. These conditions give ample opportunity for both competitive and cooperative interactions between individuals of the same and different species. Cooperative behaviour benefits the group of neighbouring microbes but comes at a fitness cost for the cooperating individuals. This creates a conflict of interest between the fitness of the individual and the fitness of the group. Individuals that defect from cooperation and therefore do not pay the cost but nevertheless benefit from the cooperative behaviour of others are called cheaters. Cooperative behaviour in the presence of cheaters constitutes altruism towards the cheaters. The aim of this review is two-fold: first, to introduce key concepts from kin selection and group selection theory that allow us to understand how cooperative behaviour can evolve in the face of cheaters; secondly, to draw attention to the conflicts of interest prevalent in biofilms yet largely ignored in the biofilm literature. Examples discussed comprise growth restraint in stationary phase as an instance of the Prisoner's Dilemma, growth restraint to allow channel formation, restraint in resource consumption or economical use of resources as altruistic behaviour, population heterogeneity as insurance against environmental changes, cooperative investment in diffusible exoenzymes, cooperation of pathogens and virulence, diffusion sensing versus quorum sensing and the inflation of signals, antibiotic resistance as collective action, and programmed cell death.
Research Articles
Quantitative analyses of Streptococcus mutans biofilms with quartz crystal microbalance, microjet impingement and confocal microscopy
- J. Kreth, E. Hagerman, K. Tam, J. Merritt, D. T. W. Wong, B. M. Wu, N. V. Myung, W. Shi, F. Qi
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- Published online by Cambridge University Press:
- 05 April 2005, pp. 277-284
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Microbial biofilm formation can be influenced by many physiological and genetic factors. The conventional microtiter plate assay provides useful but limited information about biofilm formation. With the fast expansion of the biofilm research field, there are urgent needs for more informative techniques to quantify the major parameters of a biofilm, such as adhesive strength and total biomass. It would be even more ideal if these measurements could be conducted in a real-time, non-invasive manner. In this study, we used quartz crystal microbalance (QCM) and microjet impingement (MJI) to measure total biomass and adhesive strength, respectively, of S. mutans biofilms formed under different sucrose concentrations. In conjunction with confocal laser scanning microscopy (CLSM) and the COMSTAT software, we show that sucrose concentration affects the biofilm strength, total biomass, and architecture in both qualitative and quantitative manners. Our data correlate well with previous observations about the effect of sucrose on the adherence of S. mutans to the tooth surface, and demonstrate that QCM is a useful tool for studying the kinetics of biofilm formation in real time and that MJI is a sensitive, easy-to-use device to measure the adhesive strength of a biofilm.
Review Article
Modeling biofilm complexity by including active and inert biomass and extracellular polymeric substances
- C. S. Laspidou, B. E. Rittmann
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- Published online by Cambridge University Press:
- 23 February 2005, pp. 285-291
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We present an overview of all the features of the unified multi-component cellular automaton (UMCCA) model. The UMCCA model describes quantitatively the complexity of biofilms for all biofilm components: active bacteria, inert biomass and extracellular polymeric substances (EPS). It also includes original donor substrate, two types of soluble microbial products (SMP) and oxygen. All mass balances and parameters used in UMCCA are based on our unified theory, which reconciles the apparently disparate findings about active and inert biomass, EPS and SMP. The UMCCA model captures all trends observed experimentally regarding biofilm density, namely large density differences within individual biofilm samples, with bottom layers (closest to the substratum) being as much as 5 to 10 times denser than top layers, which appear to be “fluffy” and porous. To capture this effect, the UMCCA model employs the novel idea of biofilm consolidation: the fluid over the biofilm creates pressures and vibrations that cause the biofilm to consolidate, or pack itself to a higher density over time. As a result, each biofilm compartment in the model output consolidates to a different degree that depends on the age of its biomass, which is higher for the bottom layers of the biofilm. The UMCCA model can also be used to describe biofilm mechanical properties variable in time and space, and it can be linked to finite-element software to study the behavior of biofilms under tensile or compressive stress and to perform a stress analysis of the deformed biofilm, possibly indicating where it is likely to fail or detach.
Research Articles
Detailed three-dimensional analysis of structural features of Myxococcus xanthus fruiting bodies using confocal laser scanning microscopy
- R. Lux, Y. Li, A. Lu, W. Shi
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- Published online by Cambridge University Press:
- 23 February 2005, pp. 293-303
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Myxococcus xanthus is a motile soil bacterium with complex social behaviors. Upon starvation, a developmental program is initiated that results in cellular aggregation and fruiting body formation. This process requires the exopolysaccharide (EPS) component of the extracellular matrix. With prolonged starvation, a part of the cells within a fruiting body die, while the other cells differentiate into spores. Extensive genetic and biochemical information has been generated that elucidates this interesting developmental process. Little is known, however, about the detailed three-dimensional structural features of native fruiting bodies or the EPS and distribution of live/dead cells (spores) within these structures. In this study, changes in the three-dimensional architecture of fruiting bodies and the distribution of the extracellular matrix within the fruiting bodies during the developmental process were investigated using a gfp-expressing M. xanthus strain and carbohydrate-specific lectins or monoclonal antibodies in combination with confocal laser scanning microscopy. The extracellular matrix was found to form a scaffold within the fruiting body structure. Furthermore, using a bacterial viability staining assay, the distribution of live/dead cells within fruiting bodies was examined at different times. The majority of live cells were found to localize at the outer layer of a mature fruiting body, with dead cells underneath.
Colony formation in bacteria: experiments and modeling
- M. Matsushita, F. Hiramatsu, N. Kobayashi, T. Ozawa, Y. Yamazaki, T. Matsuyama
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- 24 February 2005, pp. 305-317
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We present experimental results of colony formation in bacteria as an example of pattern formation resulting from reproduction and movement in biological populations. The bacterium Bacillus subtilis is known to exhibit at least five distinct types of colony pattern, depending on the substrate softness and nutrient concentration: diffusion-limited aggregation (DLA), compact Eden-like, dense branching morphology (DBM), concentric ring-like, and disk. We established a morphological diagram of the colony patterns, and then examined and characterized both macroscopically and microscopically how the the colonies grow. There seem to be two kinds of bacterial cells – active and inactive – and the active form drives the colony interfaces outwards. The active cells may be clearly distinguished from the inactive ones as they form the characteristic fingernail-like structure at the tips of growing branches of the DBM colony. The concentric ring-like colony is formed as a consequence of repeated alternate migration and resting of the growing interface, the cycle time for which seems to be independent of the substrate softness and nutrient concentration. So far there have been several phenomenological models proposed to qualitatively explain or reproduce the patterns observed in bacterial colonies. A few of them are reviewed here, systematically and critically, in light of our experimental results.
Review Article
The A-factor regulatory cascade that leads to morphological development and secondary metabolism in Streptomyces
- Y. Ohnishi, S. Horinouchi
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- Published online by Cambridge University Press:
- 05 April 2005, pp. 319-328
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A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) is a chemical signalling molecule, or microbial hormone, that triggers aerial mycelium formation and secondary metabolism in Streptomyces griseus. A-factor pro- duced in a growth-dependent manner switches on the transcription of adpA, encoding a transcriptional activator, by binding to ArpA, the A-factor receptor protein, which has bound to the adpA promoter, and dissociating the bound ArpA from the DNA. AdpA then activates a number of genes of various functions required for morphological development and secondary metabolism, forming an AdpA regulon. ArpA, which belongs to the TetR family, contains a helix–turn–helix DNA-binding motif in its N-terminal portion and an A-factor-binding pocket (5 Å (0.5 nm) diameter and 20 Å (2 nm) long) in its C-terminal portion, as implied by X-ray crystallography of CprB, an ArpA homologue. The ligand pocket, which can accommodate an entire A-factor-type molecule of γ-butyrolactone, is completely embedded in the C-terminal portion. Upon binding A-factor, a long helix connecting the A-factor-binding and ligand-binding domains is relocated, as a result of which the DNA-binding helix moves outside, resulting in dissociation from DNA. AdpA, which belongs to the AraC/XylS family, contains a ThiJ/PfpI/DJ-1-like dimerization domain in its N-terminal portion and an AraC/XylS-type DNA-binding domain in its C-terminal portion. For transcriptional activation, AdpA can bind to various positions with respect to the transcriptional start points of the target genes and sometimes to multiple sites. We show here how A-factor triggers secondary metabolism and morphological development in S. griseus, with emphasis on the two key transcriptional factors, ArpA and AdpA, in the A-factor regulatory cascade.
Biocomplexity in the oral cavity – the basics of structure in supragingival bacterial communities
- R. J. Palmer, P. I. Diaz, P. E. Kolenbrander
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- Published online by Cambridge University Press:
- 05 April 2005, pp. 329-335
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The human oral microbial ecosystem is one of the best characterized and highly complex bacterial communities known. It is estimated that about 600 bacterial species exist in the mouth and that 85% of those are currently known at the molecular (16 S rRNA) level. The major bacterial physiologies occurring in the oral cavity have been known for many years, and knowledge exists of the distribution of organisms in time (as plaque accumulates) and in space (different environments within the mouth). However, only rudimentary data are available on interactions between the bacterial species. It is precisely these interactions that, along with the interactions of the developing community with its human host, must drive the succession of genera that is observed to occur. In the reductionist scientific approach to studying such interactions, disrupted plaque is used to isolate single organisms, and interactions between these organisms are examined by recombining the organisms ex situ. Lessons learned from these in vitro studies can be applied to understand empirical observations made in vivo. However, this approach begins with the primary assumption that the chosen interaction does in fact occur in vivo. The accessibility and the well-characterized nature of the oral ecosystem presents an opportunity for approaching the problem from the opposite direction; one can capture a community very early in development in vivo, then apply in vitro methods to sort out the interactions within that community. This latter approach begins with a set of organisms known to interact in vivo. A combination of both approaches should yield robust microbiological data suitable for in silico modeling and analyses.
Advances in mathematical modeling of biofilm structure
- C. Picioreanu, J. B. Xavier, M. C. M. van Loosdrecht
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- Published online by Cambridge University Press:
- 28 February 2005, pp. 337-349
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Mathematical modeling of spatial biofilm structure has been in development for the past 10 years, its main goal being to derive the dynamics of biofilm structure from first-principle descriptions of the various physical, chemical and biological processes involved in biofilm formation. Early efforts described development of unrestricted monospecies consortia, often considering diffusion and reaction of a single solute species. Multi-dimensional modeling of biofilms has presently reached a stage where multi-species systems with any number of bacterial and solute species, reactions and arbitrary detachment scenarios may be readily implemented using a general-purpose software framework introduced recently. The present work presents motivations for the mathematical modeling of biofilm structure and provides an overview on major contributions to this field from pioneering efforts using cellular automata (CA) to more recent methods using the preferred individual-based modeling (IbM). Recent examples illustrate how biofilm models can be used to study the microbial ecology in: (a) development of multi-species nitrifying biofilms with anammox bacteria, (b) interspecies hydrogen transfer in anaerobic digestion methanogenic consortia, (c) competition between flock-formers and filamentous bacteria influenced by environmental conditions and its effect on morphology of activated sludge flocs, and (d) a two-species biofilm system with structured biomass describing extracellular polymeric substances (EPS) and internal storage compounds. As recent efforts from direct comparison of structure predicted by three-dimensional modeling with that observed by confocal laser scanning microscopy imaging of biofilms grown in laboratory flow cells show a good agreement of predicted structures, multi-dimensional modeling approaches presently constitute a mature and established methodology to enhance our understanding of biofilm systems.
Research Articles
Early events and pattern formation in Listeria monocytogenes biofilms
- P. Takhistov, B. George
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- 11 February 2005, pp. 351-359
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Biofilm initiation and development is a complex process that includes several major stages. This study examined the process of Listeria monocytogenes biofilm initiation and consequent spatial and physiological patterns formed in the biofilm as the result of bacterial response to a changing environment. This is a first attempt at establishing a link between the spatial organization of biofilm-associated bacteria and major stress factors such as surface bioavailability and nutrient content. Developed linear models allow evaluation of some important parameters of the surface population, such as the critical colony size and the intercolony distance in the case of diffusion-limited nutrient access. Variations in bacterial physiological patterns during biofilm initiation as a function of bacterial population density during surface colonization are reported.
Differential tolerance of Pseudomonas putida biofilm and planktonic cells to desiccation
- M. van de Mortel, W.-S. Chang, L. J. Halverson
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- 16 February 2005, pp. 361-368
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The desiccation tolerance of Pseudomonas putida strain mt-2 cells within intact unsaturated biofilms and cells cultivated in liquid media was examined. Since as soils dry there can be an increase in the solute potential and reductions in nutrient availability, we also assessed the effect of the solute and matric (low-water-content) components of the total water potential, and nutrient availability on cell survival. Biofilms were cultivated in model laboratory systems that can simulate the matric and solute components of the total water potential and the poorly mixed nature of unsaturated habitats. Desiccation stress was imposed by exposing biofilm and planktonic cells to environments with controlled relative humidities. Our results show that biofilm cells are more tolerant to air drying than are planktonic cells, and tolerance is enhanced when there is an adequate supply of nutrients available to the cells. We provide evidence suggesting that a major factor contributing to desiccation sensitivity is the matric stress itself rather than solute stress and that matric stress is inherently more stressful to P. putida than is the thermodynamically equivalent solute stress.
A simple cellular automaton model for coaggregation
- J. Wimpenny, R. Colasanti
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- Published online by Cambridge University Press:
- 01 March 2005, pp. 369-375
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Aggregation is a common phenomenon in microbiology. It manifests itself in different forms ranging from loose irregular structures such as effluent floc and marine snow to the ordered aggregates that show the reproducible patterns seen in many microbial colonies. Aggregation conveys advantages to microorganisms. These include transfer of chemical signals, exchange of genetic information, protection from adverse environmental conditions, metabolic cooperation between different species, as well as cell differentiation in some populations. Coaggregation is now recognized as a mechanism for allowing specific association between collaborating bacterial species. We describe a simple cellular automaton that illustrates structures that could form when different species interact. In particular the importance of neighbours is investigated.
A modelling study of the activity and structure of biofilms in biological reactors
- J. B. Xavier, C. Picioreanu, M. C. M. van Loosdrecht
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- Published online by Cambridge University Press:
- 01 March 2005, pp. 377-391
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In spite of the large range of morphologies observed for biofilms, there is strong experimental and theoretical evidence that the complex nature of biofilm structure dynamics is primarily a consequence of the effect of environmental conditions on biofilm development. It has been observed from the operation of industrial and laboratory-scale biofilm reactors that the structure of biofilms results from a balance of the detachment forces and the regimen of transport of a growth-limiting substrate. The overall performance of biofilm reactors is intrinsically dependent on biofilm morphology. The spatial distribution of the diverse dissolved and particulate components through the biofilm matrix and the shape of its external surface influence the rates of the occurring bioconversions, and structure also influences the stability of the biofilm in terms of resistance to mechanical stress. Individual-based modelling (IbM) of biofilms structure dynamics is used here to unify observations from the operation of biofilm reactors by simulating biofilm growth under variable detachment forces and mass transport regimens for a growth-limiting substrate. The IbM is a bottom-up approach, where the global system behaviour is derived from the local interactions of multiple elements acting independently. Transport and reaction of a solute species, local microbial growth rates and the effect of external detachment forces applied to the biofilm are modelled using differential approaches. Simulations carried out in two-dimensional space using this model illustrate a range of biofilm morphologies that emerge from different reactor operation parameters, reproducing trends observed experimentally. Comparison of multi-dimensional modelling results with those obtained using one-dimensional approaches enforces the need to use multi-dimensional modelling to predict properties that derive from the spatial biofilm structure.