ñez Group Research

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Biophysics of Bacterial Biofilms

Biofilms are complex microbial communities that grow at interfaces.  Bacteria in biofilms are phenotypically different than their planktonic (free swimming) relatives; they adapt to the communal, sessile lifestyle by expressing a specific complement of genes that allows them to optimize their motility, adhesion, and metabolism for this specialized environment.  Within these communities bacteria organize themselves to form complex architectures, differentiate to carry out distinct roles, and communicate with other cells using small molecules in ways that were once thought to be characteristic only of eukaryotes.  Biofilms are robust, dynamic, and difficult to control or destroy, which makes potential removal agents of great interest in medical, industrial, and agricultural settings.

Our interest has focused on the removal of simple bacterial biofilms by the predatory bacterium Bdellovibrio bacteriovorus. Bdellovibrios are Gram negative bacteria that consume a wide variety of other Gram negative bacteria by burrowing into the prey periplasm and breaking down the prey cytoplasm for food, digesting the prey from the inside out.

Diagram of the 2-stage life cycle of the bacterial predator Bdellovibrio bacteriovorus, which consists of a free-swimming stage spent living in water or soil, and a growth stage spent inside its prey bacterium. A Bdellovibrio predator and the killed prey cell in which it is growing is together termed a bdelloplast.

Using Atomic Force Microscopy, Occidental College professor Eileen Spain, students, and I previously demonstrated that bdellovibrios can consume simple bacterial biofilms at hydrated air-solid and air-liquid interfaces.

Exploring Predation in Bacterial Biofilms by AFM

Atomic force microscopy, or AFM, is a type of scanning probe microscopy. With AFM a very small, sharp probe is scanned across a surface, and small deflections of the probe are measured to detect changes in the topography of the surface:

With soft biological samples such as macromolecules and cells, AFM can routinely resolve structures that are tens or hundreds of nanometers in size. As optical microscopy is to vision, AFM is to Braille: because the tip touches the surface, the technique can potentially reveal no only information about shape and size, but also about tip-sample interactions involving texture, adhesion, and elasticity of the sample. AFM's major benefit is that it provides better resolution than light microsopy, and samples need not be fixed or stained, nor imaged under vacuum as for electron microscopy. Indeed, samples can be viewed in air or in liquid medium. However, AFM does have the drawback that the object to be imaged must be supported by a solid surface.

Bacteria are very difficult to image by AFM under fluid because they float away unless they are fixed in place chemically or mechanically, which can heavily modify or distort the cells of interest. However, bacterial biofilms are naturally adhered to surfaces and thus are well-suited to study by AFM.

We used AFM to study the bacterial predator Bdellovibrio bacteriovorus. The image at left shows several of the small, oblong predators consuming E.coli.

(image by Phyllis Chan, Oxy '05)

In our initial studies, we detected distinct differences in the surface of invaded and native prey cells using the deflection data in contact mode. This discovery suggested that the prey's outer membrane was physically stretched out by the predator, or that the cell's membrane potential or ion balance was breached, or that the predator made distinct changes in the chemical composition of the outer membrane.

We are exploring these possibilities using the force measurement modes of the AFM. In addition to its substantial imaging capabilities, the AFM can be used to quantitatively measure important parameters about a sample by measuring the forces exerted on the tip when it is pressed into the sample surface or retracted, allowing us to quantitatively measure the elasticity or adhesion of a surface. Recently former postdoc Dr. Megan Ferguson and Catherine Volle (MHC '06) studied the physical properties of five kinds of bacteria living in simple biofilm communities on a glass surface, in collaboration with MHC physics professor Kathy Aidala.  Notably, the biofilm-forming cells have a high cellular spring constant, indicating that they are quite stiff and hinting that stiff bacteria may preferentially colonize surfaces in the early stages of biofilm formation.  The extension curves indicate that the biofilm forming cells are coated by a soft layer of associated extracellular polymeric substances.  EPS layers are known to facilitate bacterial adhesion to surfaces during biofilm formation, and we observe them adhering strongly to the tip as it is retracted.  Pili and flagella, imaged on all but one of the biofilms, may contribute to adhesion events as well.

Using the same techniques, we probed E.coli biofilms during predation by bdellovibrios. Bdelloplasts are less elastic (i.e. have a lower turgor pressure) and are more adhesive than uninfected prey cells. Notably, these trends are consistent with the biochemical changes that are known to occur in the prey cell following infection by Bdellovibrio

These studies provide a provocative foundation for our exploration of the biophysics of bacterial biofilms.


Megan E. Núñez, Department of Chemistry, Mount Holyoke College

50 College Street, South Hadley MA 01075.

phone (413) 538-2449. fax (413) 538-2327. email menunez at mtholyoke dot edu.