- Research article
- Open Access
Holdfast spreading and thickening during Caulobacter crescentus attachment to surfaces
© Li et al.; licensee BioMed Central Ltd. 2013
Received: 2 April 2013
Accepted: 13 June 2013
Published: 19 June 2013
Adhesion to surfaces facilitates many crucial functions of microbes in their natural habitats. Thus understanding the mechanism of microbial adhesion is of broad interest to the microbiology research community.
We report a study by fluorescence imaging and atomic force microscopy on the growth in size and thickness of the holdfast of synchronized Caulobacter crescentus cells as they attach to a glass surface. We found that the holdfast undergoes a two-stage process of spreading and thickening during its morphogenesis. The holdfast first forms a thin plate on the surface. The diameter of the holdfast plate reaches its final average value of 360 nm by the cell age of ~ 30 min, while its thickness further increases until the age of ~ 60 min. Our AFM analysis indicates that the holdfast is typically thicker in the middle, with gradual falloff in thickness towards the outer edge.
We propose that the newly secreted holdfast substance is fluid-like. It has strong affinity to the surface and cures to form a plate-like holdfast capable of supporting strong and permanent adhesion.
In the environment, bacteria are predominantly attached to biotic or abiotic surfaces, where they are held by adhesive molecules at the surface of the cell envelope. Despite identification of adhesins in many bacterial species, little is known about the nature of the adhesive process from the material science point of view. In order to gain insight about the material properties of bacterial adhesins, we study the morphogenesis of the adhesive holdfast of the Gram negative bacterium Caulobacter crescentus. C. crescentus is a ubiquitous bacterium that can be found in wet soil and aquatic environments [1, 2]. Its asymmetric cell division produces a motile swarmer cell and a sessile stalked cell. The swarmer cell swims by rotating its single polar flagellum [3–6]. This mechanism allows for dispersal of the progeny cells following each division, which reduces local competition for nutrients. The swarmer cell also harbors pili, which are synthesized at the flagellar pole immediately after cell division . The stalked cell is typically attached to a surface by a holdfast found at the end of a thin, elongated extension of the cell envelope, called a stalk. The stalk is thought to increase nutrient uptake, which is particularly important in nutrient-deficient environments where molecular uptake is limited by diffusion .
The flagellum, pili, and the holdfast play important roles in surface adhesion [9–11]. Reversible adhesion occurs in swarmer cells where initial surface interactions are mediated by the flagellum and pili . Contact of the flagellum and pili with a surface increases the load on the flagellum motor, halting flagellum rotation and triggering just-in-time deployment of holdfast from the flagellar pole. The attached cell subsequently develops into a stalked cell with elongation of a thin stalk from the pole bearing the holdfast. In cells that do not contact a surface, holdfast synthesis is regulated by the developmental program and occurs in the late swarmer stage [11, 12]. There has not been much study with respect to possible differences between these two pathways, since the contact-triggered C. crescentus adhesion pathway has only been discovered recently .
The C. crescentus holdfast is a complex of polysaccharides and proteins required for adhesion to surfaces with impressive strength [9, 13–15]. The fluorescently labeled lectin fluorescein isothiocyanate-wheat germ agglutinin (FITC-WGA), which binds to oligomers of N-acetylglucosamine (GlcNac or NAG), binds specifically to the holdfast, indicating that the holdfast contains NAG . Furthermore, the holdfast is sensitive to treatment with lysozyme, which cleaves NAG polymers [13, 16]. Mutants that cannot be stained with FITC-WGA are unable to form irreversible surface adhesion .
In this paper, we used fluorescence microscopy and atomic force microscopy to study holdfast growth of cells attached to a surface. We show that the holdfast undergoes a two-stage process of spreading and thickening during its morphogenesis. Based on the observed holdfast growth characteristics, we propose that the newly secreted holdfast material is a fluid-like substance that cures to form a plate-like holdfast capable of supporting strong and permanent adhesion.
Strain and synchronization
Wild-type C. crescentus strain CB15 was cultured in a peptone-yeast extract (PYE) medium  at 30°C. Synchronized swarmer cells were obtained using a plate releasing technique [12, 17]. Unless specified, the synchronized cells were harvested 5 min or less after cell division. The age variance of these cells, with time counted from separation and release of the swarmer cell, was within 5 min. In selected experiments, young swarmer cells were also synchronized to a narrower range of within 1 min in age in order to best resolve the early stages of holdfast development.
Fluorescence labeling of holdfasts
Holdfasts were labeled as described previously . A drop of synchronized swarmer cells was placed on a coverslip for 5 min, allowing some swarmer cells to attach to the glass surface. For the study of cells younger than 6.5 min, incubation time was reduced to 1 min. The unattached cells were rinsed off gently with fresh PYE and the cells attached to the coverslip were then grown at 30°C for various lengths of time. After growth, the coverslip was rinsed with water to remove nutrients. Cells were labeled with fluorescein-conjugated WGA solution on ice for various amounts of time, supplemented with 0.05% (w/v) sodium azide to stop cell growth during the labeling. The concentration of the fluorescein-WGA varied from 0.02 to 1 mg/ml. After labeling, the coverslip was rinsed with the sodium azide solution three times and an anti-photobleaching solution was added to the coverslip prior to fluorescence microscopy. The anti-bleaching solution contained 20 μg/ml catalase, 0.5 mg/ml glucose, 0.1 mg/ml glucose oxidase, and 0.25 vol% ß-mercaptoethanol .
A Nikon Eclipse E800 epifluorescence microscope with a 100 × oil immersion objective lens (Plan Apo) was used to image the fluorescently labeled holdfast. A highly sensitive and linear CoolSnap camera was used to record the fluorescence images of holdfasts, controlled by MetaMorph (Universal Imaging, PA) software. The attached cells were first brought into focus under phase contrast setting for easy location of the cells. Then the holdfasts were observed under fluorescence mode with fine adjustment of focus. Consecutive fluorescence images were taken with 0.1 s exposure time while manually adjusting the focus with the fine adjustment knob. Optimal focus was achieved within ten attempts. The image of the 10th exposure was used to obtain the fluorescence intensities of holdfasts.
Measurement of fluorescence intensity
To measure the integrated fluorescence intensity, a circle larger than the holdfast image was drawn using the imaging software and the intensity was integrated over all the pixels inside the circle. The sum was then subtracted by the integrated background intensity of a nearby circle of the same size to obtain the integrated intensity of the holdfast. This method eliminates background intensity from the camera noise and from dye molecules adsorbed on the glass surface. The net integrated fluorescence intensity of holdfasts was measured for over 500 cells older than 7.5 min in age per time point. The fluorescence images of most holdfasts were sufficiently bright and their intensities were measured by an automated routine using the commercial software Matlab (Mathworks, Natick, MA, USA). A small sub-population of holdfasts were too dim to be recognized by the Matlab program and their intensities were determined individually by the integrated intensity function in MetaMorph. For cells younger than 6.5 min, fluorescence intensities of almost all holdfasts were too weak to be recognized by the Matlab program. Instead, about 100 holdfasts at each chosen age were measured individually using MetaMorph.
Selection of experimental condition for quantitative fluorescence analysis
We used the following method to determine proper fluorescein-WGA labeling conditions. Synchronized swarmer cells were allowed to quickly attach to a glass microscope coverslip. The unattached cells were washed away. The attached cells were incubated for 27.5 min at 30°C to ensure formation of holdfasts. We then measured average intensity of those holdfasts labeled with 20, 100, and 500 μg/ml fluorescein-WGA for 15 min and average intensity of holdfasts labeled with 100 μg/ml fluorescein-WGA for 5, 10, 15 and 20 min in order to determine the dependence of the average integrated fluorescence intensity on dye concentration and incubation time. We found that the integrated fluorescence intensity was not sensitive to the lectin concentration or labeling time within these ranges, suggesting saturation of dye labeling under these experimental conditions. We concluded that performing labeling experiments within these ranges allows robust comparison of holdfast size even with somewhat variable amounts of dye and incubation time, as long as they were above 20 μg/ml and 5 min, respectively. For all subsequent experiments, we labeled the holdfasts with 100 μg/ml lectin for 15 min.
Atomic force microscopy (AFM)
In order to obtain a clean surface as a substrate for AFM imaging, glass coverslips were soaked in a solution of 6 % (w/v) Nochromix (GODAX Laboratories, Inc.) in concentrated H2SO4 for 1 hour and then rinsed thoroughly with deionized water. A drop of culture containing synchronized swarmer cells was placed on a clean coverslip for 5 min. The unattached cells were rinsed off with oxygenated fresh PYE and the attached cells were then grown at 30 °C over various time intervals to allow for holdfast growth. The coverslip was then blow-dried gently with compressed N2 gas so that the attached cells fell over to the side, getting stuck and dried onto the glass surface. The dried cells and their holdfasts, also dried on the glass surface, were imaged using a Nanoscope IIIa Dimension 3100 (Digital Instruments, Santa Barbara, CA) atomic force microscope using contact mode in air.
Distribution of holdfast fluorescence intensity at various ages
The holdfast spreads to a thin plate at the attachment site
The holdfast undergoes a two-stage process of spreading and thickening
The distinct time course for the spreading and thickening of a new holdfast offers important insights into the material properties of the holdfast. Newly secreted holdfast material appears to behave as a viscous fluid, which spreads quickly over a flat solid surface. The physics phenomenon is akin to what is often called “wetting” [19, 20], typically a process during which a liquid drop spreads over a solid surface in the ambient environment. For this analogy to be valid the holdfast material must not mix with the growth medium and there ought be significant surface tension at the holdfast/medium interface. In addition, the holdfast must have strong affinity for the surface. All these conditions appear to have been met, leading to the adhesion characteristics observed.
The AFM images and particularly the height scan as illustrated in Figure 5b offer further insights on the curing process of newly secreted holdfast material. Because holdfasts are thin and the contact angle at the edge of the holdfast is small, the size of the holdfast does not appear to be caused by balancing the forces of line tension at the contact edge and the weight of the spreading liquid drop. Instead, the holdfast size may be dictated by the rate of gelation of the holdfast. Once the first thin layer is cured, the additional secretion might spread over the gelled disk and cures in comparable or even shorter amounts of time, thus continually thickening the gelled holdfast until the secretion stops. The fact that the holdfast stops spreading but continues to thicken indicates that some kind of molecular transformation takes place faster than the time for the new secretion to spread past the footprint of the holdfast cured from the initial spread. Caulobacter cells can adhere strongly to a wide variety of surfaces, including glass, plastics, and metals [10, 13]. The non-specific nature of these strong interactions implies that they are non-covalent and most likely attributable to van der Waals forces [21, 22]. The major component of the holdfast, polymers of N-acetylglucosamine, may be well suited as the base material for a wet adhesive. It appears to produce strong molecular interactions with many solid materials due to non-specific interactions; it does not disperse in an aqueous environment upon secretion due to a high degree of crosslinking. Unfortunately, the detailed composition of the holdfast remains unknown and we know nothing about the processes that triggers the curing of newly secreted holdfast material.
Adhesives have a broad range of biomedical applications, from denture to surgical suture. A good bio-adhesive must be fast to cure, waterproof, and resilient once bonded with a range of different materials. A synthetic adhesive often relies on catalytic reactions to cure, such as in an epoxy-resin mixture. The curing of adhesive mixtures for medical and dental applications is typically triggered by UV light, which conveniently triggers crosslinking reactions at the desirable site. Most natural biological adhesins, such as the holdfasts secreted by Caulobacter crescentus and several species of alphaproteobacteria [23–25], adhere to solid surfaces under normal aqueous conditions. This important property naturally selected during the course of evolution may soon be harnessed for biomedical applications.
This work was supported by the National Institutes of Health Grants GM077648 and GM102841 to Y.V.B. and the National Science Foundation Award PHY 1058375 to J.X.T.
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