Bacterial colony from two-dimensional division to three-dimensional development

Abstract

On agar surface, bacterial daughter cells form a 4-cell array after the first two rounds of division, and this phenomenon has been previously attributed to a balancing of interactions among the daughter bacteria and the underneath agar. We studied further the organization and development of colony after additional generations. By confocal laser scanning microscopy and real-time imaging, we observed that bacterial cells were able to self-organize and resulted in a near circular micro-colony consisting of monolayer cells. After continuous dividing, bacteria transited from two-dimensional expansion into three-dimensional growth and formed two to multi-layers in the center but retained a monolayer in the outer ring of the circular colony. The transverse width of this outer ring appeared to be approximately constant once the micro-colony reached a certain age. This observation supports the notion that balanced interplays of the forces involved lead to a gross morphology as the bacteria divide into offspring on agar surface. In this case, the result is due to a balance between the expansion force of the dividing bacteria, the non-covalent force among bacterial offspring and that between bacteria and substratum.

Figure 1. Observations of EHEC daughter cells' arrangements during the early division.

EHEC O157:H7 was grown on a 1.5% LB-agar-coated slide (immersed in LB medium) and the early bacterial divisions were observed by time-lapsed microscopy. Daughter cells slid side-by-side after the first fission and then formed a 4-cell array after the 2^nd round of division. The arrow indicates the irregular arrangement of octomeric daughter cells that are to grow into a nearly circular micro-colony after a few more cycles of division. Scale bar: 5 µm.

Figure 2. Micro-colonies observed by different microscopic imaging systems.

GFP-expressing EHEC was cultivated as in Fig. 1. Micro-colony was observed: (A) on the xy plane with phase-contrast microscopy; (B) on the xy plane with CLSM; (C) on the xz plane along the diameter-line “D” with CLSM (sectioned at thickness of 0.5 µm). Images were taken after 4 h of cultivation. (D–F) Individual images were taken as that to (A–C), respectively, except for cultivation for 5 h. Note: a light outer ring and a dense center core concentrically seen in (D) are marked; the radius of the denser center core and that of the whole micro-colony are labeled “r” and “R”, respectively, in (F) and the difference between r and R is W_m, the transverse width of a monolayer. Arrows indicate representative cells oriented approximately perpendicular to the radial direction of the micro-colony.

Figure 3. Time-lapse image analysis of the structures in the 3^rd dimension of a developing micro-colony.

A growing bacterial micro-colony similar to that in Fig. 2 was followed by using a confocal laser scanning microscope (sectioned with a thickness of 0.05 µm). (A, C) Projected images at different time points for the xz plane after cross-sectioning along the D line of the micro-colony. r: the radius of the center core; W_m, the transverse width of the outermost monolayer. (B, D) A series of images from the phase contrast microscopy. Arrows indicate the spaces of the monolayer rings. Scale of a gird in A and C: 10 µm.

Figure 4. Imaging the multi-layer structure of micro-colony.

Imaging was carried out as described earlier for the case of Fig. 2 that the sectioning thickness of CLSM was set at 0.5 µm. All phase-contrast images were taken at the same scale to show the change in sizes of growing micro-colonies: (A) 5 h; (B) 6 h; (C) 7 h. To the right of individual phase-contrast images are the xz plane along the D line of the micro-colony. In each vertical panel, the images were taken for the middle, upper and bottom parts of the micro-colony, respectively. Note: the magnification of the confocal florescence images were 3.5 times that of the phase contrast images.

Figure 5. Characteristics summarized for the developing micro-colony.

(A) Measured widths of the outermost monolayer during micro-colony formation. Measurements of W_m values of individual micro-colonies were carried out similarly to that described in Fig. 5. Every single spot represents a mean of two W_m values of a monolayer ring measured from images of the xz plane. Horizontal line marks the average of each measurement group. Three asterisked pairs indicate that there is a significant difference between the groups (p<0.0001 by t-test). (B) Illustration of a micro-colony development by viewing at different planes. Even though the diameter and layers of the bacterial micro-colony were increasingly expanding, the average width (W_m) of the outermost monolayer reached a constant value after approximately 6 h of growth. The red arrow indicates that the constant outward force per unit length. Note: the boundaries and intensities between layers are not as sharp as illustrated, particularly those beyond the 2^nd/3^rd layers, and scales are not proportionally represented.

Figure 6. Orientation angle analysis of bacteria lying at the front edge of micro-colonies.

Bacterial micro-colonies (n = 14), cultivated and imaged as shown in Fig. 2A, of roughly circular shape were used for analysis. (A) Illustration of a representative roughly circular-shaped micro-colony with the contours of individual bacteria shown. Three concentric circles were drawn to define the center of the micro-colony, the first loop that contains the outermost cells, and the second loop that is used to compare with the first loop. The two loops have the same width (2 µm). The acute angle (θ) between the cell orientation, determined from the end-to-end line segment (double-headed arrow), and the radial direction (blue line) was measured. (B) Distribution of the bacterial orientation angles. Upper panel: cells in the outermost loop 1. Order of the polynomial fitting is 2 and R square is 0.876. Lower panel: bacteria in the second loop. Order of the polynomial fitting is 2 and R square is 0.559. (C) Comparison of the percentages of bacteria in micro-colonies with orientation angles in the range of 66° to 90° between the outermost loop 1 and the one next to it (loop 2). Three asterisks mark a significant difference (p<0.0005 by t-test) when the two groups of 66°–90° were compared.

Figure 7. Model for formation of tangential orientation when outermost bacteria receiving a torque.

(A) A likely consequence of a cell in radial orientation. Note, the limited contact surface provided by a and a torque exerted by b. (B) A tangentially positioned cell a before and after receiving multiple torques that cancel out each other.

Figure 8. Interactions of bacteria to bacteria and bacteria to substrate surface as revealed by perturbing the formation of micro-colonies.

Bacterial divisions and development into micro-colonies were carried out on a layer of thin agar immersed in LB with a coverslip-sandwiched chamber as that in Fig. 2. The chamber was lightly tapped and responses of the bacteria were immediately followed by time-lapsed microscopy. Arrowheads indicate those bacteria stayed attached to the surface but turning and vibrating could be seen with many of these cells. Arrow highlights bacteria behaving as a group that restructured their gross morphology, from a spindle-like structure (A) transiting into a near spherical shape (C), and re-positioned themselves actively (see Movie S1 in Data S1). Note: no apparent bacterial division could be seen within this short period of 6 seconds. Scale bar: 10 µm.