▪ Abstract Biofilms can be defined as communities of microorganisms attached to a surface. It is clear that microorganisms undergo profound changes during their transition from planktonic (free-swimming) organisms to cells that are part of a complex, surface-attached community. These changes are reflected in the new phenotypic characteristics developed by biofilm bacteria and occur in response to a variety of environmental signals. Recent genetic and molecular approaches used to study bacterial and fungal biofilms have identified genes and regulatory circuits important for initial cell-surface interactions, biofilm maturation, and the return of biofilm microorganisms to a planktonic mode of growth. Studies to date suggest that the planktonic-biofilm transition is a complex and highly regulated process. The results reviewed in this article indicate that the formation of biofilms serves as a new model system for the study of microbial development.

Biofilm Formation as Microbial Development

Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.

Microbial Biofilms: from Ecology to Molecular Genetics

Populations of surface-attached microorganisms comprising either single or multiple species are commonly referred to as biofilms. Using a simple assay for the initiation of biofilm formation (e.g. attachment to an abiotic surface) by Pseudomonas fluorescens strain WCS365, we have shown that: (i) P. fluorescens can form biofilms on an abiotic surface when grown on a range of nutrients; (ii) protein synthesis is required for the early events of biofilm formation; (iii) one (or more) extracytoplasmic protein plays a role in interactions with an abiotic surface; (iv) the osmolarity of the medium affects the ability of the cell to form biofilms. We have isolated transposon mutants defective for the initiation of biofilm formation, which we term surface attachment defective (sad). Molecular analysis of the sad mutants revealed that the ClpP protein (a component of the cytoplasmic Clp protease) participates in biofilm formation in this organism. Our genetic analyses suggest that biofilm formation can proceed via multiple, convergent signalling pathways, which are regulated by various environmental signals. Finally, of the 24 sad mutants analysed in this study, only three had defects in genes of known function. This result suggests that our screen is uncovering novel aspects of bacterial physiology.

Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis

Gram-negative cell walls are strong enough to withstand ;3 atm of turgor pressure (40), tough enough to endure extreme temperatures and pHs (e.g., Thiobacillus ferrooxidans grows at ap H of’1.5) and elastic enough to be capable of expanding several times their normal surface area (41). Strong, tough, and elasti c...t hegram-negative cell wall is a remarkable structure which protects the contents of the cell and which has stood the test of time for many, many years. Presumably, these three descriptive traits, have much to do with the tremendous success gram-negative bacteria have had as a life-form on our planet; members of the domain Bacteria inhabit almost all imaginable habitats except those excruciatingly extreme environments in which (some) members of the domain Archaea thrive. Molecular biological methods have not yet given scientists a precise historical record of the origin of gram-negative bacteria, but ancient stromatolites containing fossilized remains of cyanobacterium-like prokaryotes date back to the Archean eon. Over such extraordinary periods of time (much of it when no other life existed), we can imagine that random mutation, selection, and the slowly but ever-changing global

Structures of Gram-Negative Cell Walls and Their Derived Membrane Vesicles

https://www.cell.com/trends/microbiology/pdf/S0966-842X(01)02012-1.pdf

The biofilm matrix – an immobilized but dynamic microbial environment

The surfaces of bacteria are highly interactive with their environment. Whether the bacterium is Gram-negative or Gram-positive, most surfaces are charged at neutral pH because of the ionization of the reactive chemical groups which stud them. Since prokaryotes have a high surface area-to-volume ratio, this can have surprising ramifications. For example, many bacteria can concentrate dilute environmental metals on their surfaces and initiate the development of fine-grained minerals. In natural environments, it is not unusual to find such bacteria closely associated with the minerals which they have helped develop. Bacteria can be free-living (planktonic), but in most natural ecosystems they prefer to grow on interfaces as biofilms; supposedly to take advantage of the nutrient concentrative effect of the interface, although there must also be gained some protective value against predators and toxic agents. Using a Pseudomonas aeruginosa model system, we have determined that lipopolysaccharide is important in the initial attachment of this Gram-negative bacterium to interfaces and that this surface moiety subtly changes during biofilm formation. Using this same model system, we have also discovered that there is a natural tendency for Gram-negative bacteria to concentrate and package periplasmic components into membrane vesicles which bleb-off the surface. Since some of these components (e.g., peptidoglycan hydrolases) can degrade other surrounding cells, the vesicles could be predatory; i.e., a natural system by which neighboring bacteria are targeted and lysed, thereby liberating additional nutrients to the microbial community. This obviously would be of benefit to vesicle-producing bacteria living in biofilms containing mixed microbial populations.

Interactions between biofilms and the environment

Summary: Pseudomonas aeruginosa PAO1 possesses two distinct lipopolysaccharide (LPS) O-polysaccharide species, A- and B-band LPS, the relative expression of which appears to be under environmental control. In an attempt to identify the influence these LPS types have on surface characteristics and adhesion, we examined the surface hydrophobicity and surface charge of P. aeruginosa PAO1 (O5 serotype) and its isogenic LPS derivatives which possessed A+B−, A+B− and A−B− LPS. The surface characteristics of the strains affected their ability to adhere to hydrophilic (glass) and hydrophobic (polystyrene) surfaces. Cells possessing only A-band LPS demonstrated the highest surface hydrophobicity, followed by the strain lacking both A- and B-band LPS. The presence of B-band LPS resulted in a more hydrophilic surface. Strains lacking B-band LPS (A+B− and A−B−) had more electronegative surfaces than those possessing B-band LPS (A+B+ and A−B−), with cells lacking both A- and B-band LPS showing the highest surface electronegativity. These data suggest that the main surface-charge-determining groups reside in the core region of the LPS molecule. Cells with the lowest surface hydrophobicity and lowest surface charge (A−B−, A−B+) adhered to glass the most efficiently, implying a role for electrostatic interaction, whereas adhesion to polystyrene mirrored the relative hydrophobicities of the strains (A+B−>A+B−>A−B−>A+B+). It is postulated that phenotypic variation in the relative expression of A- and B-band LPS may be a mechanism by which P. aeruginosa can alter its overall surface characteristics in such a way as to influence adhesion and favour survival.

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Stephen A. Makin

Papers

Interactions between biofilms and the environment

The influence of A-band and B-band lipopolysaccharide on the surface characteristics and adhesion of Pseudomonas aeruginosa to surfaces