Paenibacillus dendritiformis bacterial colony growth depends on surfactant but not on bacterial motion

Transcription

1 JB Accepts, published online ahead of print on July J. Bacteriol. doi:./jb.- Copyright, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Paenibacillus dendritiformis bacterial colony growth depends on surfactant but not on bacterial motion Running title: Colony growth and microscopic bacterial motion Avraham Be er *, Rachel S. Smith, H. P. Zhang, E. L. Florin, Shelley M. Payne, and Harry L. Swinney Center for Nonlinear Dynamics and Department of Physics, University of Texas at Austin, Austin, Texas ; Section for Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas. Most research on growing bacterial colonies on agar plates has concerned the effect of genetic or morphotype variation. Some studies have indicated a correlation between microscopic bacterial motion and macroscopic colonial expansion, especially for swarming strains, but no measurements have been performed on a single strain to relate the microscopic to the macroscopic scales. We examine here for a single strain (Paenibacillus dendritiformis Type T; tip- splitting) both the macroscopic growth of colonies and the microscopic bacterial motion within the colonies. Our multi-scale measurements for a variety of growth conditions reveal that motion on the microscopic scale and colonial growth are largely independent. Instead, the growth of the colony is strongly affected by the availability of surfactant that reduces surface tension. * Corresponding author. Mailing address: Center for Nonlinear Dynamics, University of Texas at Austin, Dean Keeton and Speedway, RLM., Austin TX. Phone: () -. Fax: () -. address: beerav@gmail.com. Downloaded from on March, by guest

2 INTRODUCTION Bacteria are able to colonize many different surfaces through collective behavior such as swarming and biofilm formation. Studies of such behavior (,,,) have revealed cooperative phenomena on both microscopic and colonial scales (,,,,), including production of extracellular "lubricant-wetting" fluid for movement on medium and hard surfaces (,,), chemical signaling such as quorum sensing and chemotactic signaling (,,), and the secretion of inhibiting and killing factors (,,,,,). Research has suggested possible links between the microscopic behavior of a colony and the rate at which a colony expands (,,,). For Pseudomonas aeruginosa, increased reversal rates of flagella lead to hyperswarming (a larger colony size) (). Similar flagellar modulation affects Escherichia coli (), where, if the bacteria never tumble (flagella rotate counter-clockwise only) or only tumble (flagella rotate clockwise only), the final colony size is much smaller than when the bacteria both swim and tumble. For Rhizobium etli, a correlation has been observed between the microscopic swarming motion and the expansion of the colony, and an AHL molecule has been found to be a swarming regulator as well as a biosurfactant that controls surface activity (). These studies suggest a correlation between the microscopic activity and colonial expansion; however, a mutation may be pleiotropic, affecting both motility and surfactant production. Further, there may be additional, unidentified differences between mutant and wild type strains. As an example, for Bacillus subtilis the failure of laboratory strains to swarm is caused by a mutation in a gene (sfp) needed for surfactin synthesis and a mutation(s) in an additional unknown gene(s) (). Experiments that avoid this ambiguity by studying the response of a single strain exposed to changing physical environments have not been performed. Further, except for measurements of the size of an expanding colony as a function of time (,), no detailed time development studies of a growing bacterial colony have been reported. Here we expose a single bacterial strain, Paenibacillus dendritiformis (type T; tipsplitting) (), to different substrate hardness, nutrition levels, and surfactant Downloaded from on March, by guest

3 concentrations to identify the parameters that determine colonial growth. P. dendritiformis is a Gram-positive rod shaped ( m x m) bacteria that swim on top of the agar gel in a thin layer (a few micrometers thick) of fluid, presumably secreted by the bacteria. The bacteria develop complex colonial (bush-like) branching patterns that are sensitive to small changes in the environment when the bacteria are grown on nutrient limited (low peptone levels; on the order of g/l) surfaces (). The colonies grow slowly (. mm/h) so the microscopic motion can be followed with a microscope for about min without a need to move the field of view. Also, this strain shows swarming-like microscopic motion where the bacteria move collectively in the form of whirls and jets. This makes these bacteria well suited for studying simultaneously of the development of a colony and the internal structure of branches. We have constructed a novel set-up to observe ten growing P. dendritiformis colonies in each experiment, and complementary microscopic measurements are made of the velocity field of individual or small groups of cells of bacteria within the colonies. Specifically, we measure the bacterial speed, which is the average of the magnitude of the velocity vectors for the bacteria in a region near the edge of a growing colony, and the tip velocity, which is the speed of the moving growth front at the edge of a colony. We also quantify the collective bacterial motion within the colonies by computing spatial and temporal velocity autocorrelation functions. MATERIALS AND METHODS Strain and growth media: Paenibacillus dendritiformis (Type T; tip-splitting) bacteria () are stored at - C in Luria Broth (LB) (Sigma) with % (wt/vol) glycerol. Frozen stock was used to inoculate LB. After growing for hours at C with shaking, the culture reached an OD of., corresponding to approximately measured by counting colonies inoculated on LB plates after culture dilution. bacteria/ml, The peptone nutrient medium contained NaCl ( g/l), K HPO ( g/l), and Bacto Peptone (- g/l). Difco Agar (Becton Dickinson) was added at concentrations.-.% Downloaded from on March, by guest

4 (wt/vol). In some experiments a non-ionic commercial surfactant, Brij (Sigma), was added in various concentrations (-.% (wt/vol)) to the agar medium prior to autoclaving. Twelve ml of dissolved agar was poured into each. cm diameter Petri dish, which was dried for days at C and % humidity until the weight of the plate decreased by g to a final weight of about g. This protocol ensures reproducible results. Growth pattern experiments: The agar plates are inoculated by placing l droplets of the culture on the surface. The plates are mounted on a rotating stage inside a m chamber maintained at.. C and % humidity (see Fig. (SI) of ()). The rotating stage system allows monitoring of the growth development of ten plates simultaneously. The stage is controlled by a stepper motor that stops sequentially for each bacterial colony to be imaged. A rotation period of h is sufficiently short to capture the growth of the colony, the tips of which move typically. mm/h (~. m/s). The reproducibility of positioning of the agar plates is m, allowing successive images of a given colony to be subtracted to determine growth patterns. Images are obtained with a Megapixel Nikon D camera with a mm lens, as in (). For plates imaged only at a single point in time, colonies are stained with.% Coomassie Brilliant Blue to obtain higher contrast images than those obtained using scattered light. The stain solution consists of % methanol, % water, % acetic acid (. M) and g/l Brilliant Blue in a ml solution (poured on each plate). This stain kills the bacteria but leaves the colony blue on a pale agar background. A similar solution that lacks the g/l Brilliant Blue is then used to distain the agar. Microscopic measurements: An optical microscope (Olympus IX) equipped with a LD X Phase contrast (PH) objective lens is used to follow the microscopic motion. The microscope is placed in a temperature and humidity controlled environment. A digital camera captures the microscopic motion at a rate of frames per second and a spatial resolution of pixels. Images are taken for s periods, resulting in images in a sequence. Downloaded from on March, by guest

5 In order to quantify the bacterial motion at the microscopic level we use particle image velocimetry (,,,), which uses cross correlations over small interrogation regions between a pair of consecutive images to find particle displacements over known time intervals to obtain -dimensional local velocities V ( x, y). The resulting velocities are interpolated to a uniform regular grid by a cubic spline interpolation. The velocity data are used to compute vorticity ( local rotation rate), which is defined as the curl of the velocity field, ( x, y) V ( x, y). Velocity (and vorticity) fields are obtained on a grid with a spatial resolution of. m with % (and %) RMS errors. We calculate the spatiotemporal velocity correlation function for the microscopic velocity components V x and V y, which are respectively along the direction of colony expansion and perpendicular to the expansion. The correlation function has the form Vi ( x, y, t ) Vi ( x x, y y, t t ) Ci ( x, y, t ), where... means V ( x, y, t ) V ( x, y, t ) i i average over both space x, ) and time t, and i = x or y; the spatial averaging is done ( y over the entire region near (within ~ m) the edge of a growing tip. We will call the velocity autocorrelation function x, y, ) the whirl-correlation to distinguish C i ( t it from the relationship between the microscopic and macroscopic motions, which will be referred to as correlation. RESULTS We examine the effect of nutrient level and substrate rigidity on growth of P. dendritiformis colonies and on the motion of the bacteria within the colonies. Growth at intermediate nutrient level ( g/l peptone) for hard gel (.% (wt/vol) agar): During the first h, the bacteria grow only inside the small circle of the inoculation. Then the colony starts to expand, and a branched pattern develops (Fig. A). The velocity of the growing envelope (taken as a circle that just touches the fastest Downloaded from on March, by guest

6 growing tips) is constant, as can be seen in Fig. D (see also Movie of SI of ()). By subtracting consecutive movie frames, we found that the growth of a colony is limited to an active zone at the tips of the branches; that is, branches grow forward and not to the side (Fig. C). Thus a branch s width is already determined as it starts to grow. Regions only m back from the growing tips stop growing, even though they may have ample space available for growth on the side of a growing tip. A close look near a colony s growing tip reveals that bacteria inhabit three well-defined regions (Fig. B). Region III is very close to the growing front and contains bacteria in three (and sometimes more) layers (z-axis). The width of Region III is typically m, and this width remains constant as a colony grows from less than mm to more than mm in diameter; this suggests that the number of active bacteria per unit area is fairly constant as the colony grows. Bacteria in Region III are active, reproducing, and moving in a collective swarming-like motion in the form of whirls and jets, as illustrated by Movie of the Supplementary Information (SI). Snapshots of the velocity and vorticity fields are shown in Figs. A and C, respectively. The whirls and jets have size ~ m and last a few seconds. Region II (about m in width) is slightly more interior and is composed of layers of bacteria; these bacteria also move in whirls and jets, but the speeds of the individual bacteria are about half those in Region III (see Fig. D). The swimming speeds of P. dendritiformis bacteria in rich liquid media are typically m/s, which is also slow compared to the swimming speeds other bacteria. Region I, typically m or further from a colony s front, contains bacteria in a single layer (see inset of Fig. B); these bacteria hardly move at all (see Fig. D). The average size of bacteria in Region I is.. m, while the bacteria in Regions II and III range from to m in length. This suggests that reproduction is limited to the regions with multiple layers. Region I contains spores; the concentration of spores relative to active bacteria increases with increasing distance from a colony s edge. Downloaded from on March, by guest

7 Effect of nutrient level on macroscopic and microscopic motion: To explore environmental affects on correlations between microscopic and macroscopic motions, colonies were grown for various nutrient levels, keeping the hardness of the substrate constant (at.% (wt/vol) agar). In each case the velocity of the growing envelope was observed to be constant as a function of time. The three distinct regions observed for g/l peptone (Fig. B) were also found at other nutrient levels. For small initial peptone level, an increase in peptone concentration results in an increase in the envelope velocity (Fig. A), indicating that the growth is food limited, but the tip velocity reaches a maximum at g/l peptone, indicating that another factor limits colony growth form this level on. However, the average speed of individual bacteria continues to increase with increasing peptone levels above g/l, and at g/l the speed of the bacteria reaches values three times as large as at g/l (Fig. B). Thus in this range of nutrition the growth speed of the colony is clearly independent of the microscopic speed of the bacteria. Similarly, for a softer gel (.% (wt/vol) agar), an increase in the peptone concentration from a small initial value results in an increase in the envelope velocity (Fig. A), indicating again that the growth is food limited. However, the tip velocity saturates in this case at much higher peptone level (~ g/l). Interestingly, the increase in the average speed of the bacteria saturates much earlier at a peptone level of g/l while the tip keeps on growing faster with higher peptone levels. The striking contrast in the behavior for the.% (wt/vol) and.% (wt/vol) agar concentrations is summarized in the plot of tip velocity vs. bacterial speed (Fig. B). The results displayed in Fig. B demonstrate dramatically that the colonial growth rate does not depend on the average speed of the individual bacteria. Bacterial speed and colonial growth rate behave rather as independent parameters, coupled at lower concentrations by the availability of food. Figure B shows in addition that there is a qualitative difference in the response of the colony to different agar concentrations. On soft agar, the average speed of the bacteria reaches a maximum, while hard agar limits the colonial growth. Effect of the agar concentration on the macro/micro motion: To investigate the transition from conditions under which the maximal speed of the bacteria is limited to Downloaded from on March, by guest

8 conditions under which the tip growth velocity is limited, we varied the agar concentration in the range from.% to.% (wt/vol) while keeping the peptone level constant (for different peptone levels:,, and g/l). For soft gels, Region III (where bacteria live in layers) is larger than for hard gels; for example, for a gel with.% (wt/vol) agar, the width of Region III was about mm, compared to. mm for a gel with.% (wt/vol) agar. With increasing agar concentration, the tip velocity of a colony increases until it reaches a maximum at about.% (wt/vol) agar concentration; with further increase in agar concentration the tip velocity decreases rapidly (Fig. A). The average speed of individual bacteria shows a similar dependence on agar concentration (Fig. B), but the speed maximum occurs at a lower agar concentration (about.% (wt/vol)) than the maximum in tip velocity. The consequence of the different locations of the maxima as a function of agar concentration is illustrated by a graph of tip velocity as a function of bacterial speed (Fig. C). Both respond to the changing agar hardness but in a slightly different way, indicating again that the average bacterial speed does not drive colonial growth. Whirls, jets, and collective micro-motion: Since the tip motion seems not to be determined by the average speed of the bacteria, we investigated whether it depends instead on the collective motion of bacteria (i.e., whirls and jets, Fig. C). The spatiotemporal whirl-correlation was determined in the active region (Region III), as described in Materials and Methods. The correlation function for both V x and V y gave similar results; the one for V x is shown in Fig.. A cut made through the center of the plot is shown in Fig. B; the correlation length was defined to be the width of the peak at correlation=.. The whirl correlation length increases approximately linearly with bacterial speed (Fig. A), but the relationship between tip velocity and the whirl correlation length (Fig. B) is similar to the non-monotonic relation found between tip velocity and bacterial speed (Fig. C). Similar results were obtained whether the whirlcorrelation length was taken to be the distance between the anti-nodes. Thus we conclude that the tip growth is also not driven by the collective motion of bacteria in whirls and jets. Downloaded from on March, by guest

9 Effect of surfactant on the motion: Another possible mechanism that could drive tip growth is the reduction of surface tension by a surfactant, a mechanism that is independent of the microscopic motion of the bacteria and requires only the production of surfactant. We examined this by adding a non-ionic surfactant, Brij, when preparing the gel. We found that the tip velocity increased by a factor of. when the surfactant concentration increased from % to a final concentration of.% (wt/vol), while the average speed of the bacteria remained essentially constant (Fig. ). Further, the added surfactant did not affect the whirl-correlation length. Brij added at various concentrations (-.% wt/vol) to bacterial cultures grown in both poor and rich (LB) shaken liquid media did not affect the bacterial growth. Together, these results suggest strongly that tip growth and thus colonial growth in P. dendritiformis (type T; tip-splitting) is mainly controlled by the production of surfactant molecules. Downloaded from on March, by guest

10 DISCUSSION Rather than examining different bacterial strains, we have studied a single strain that was exposed to various nutrient levels, agar hardness, and surfactant concentrations. The use of the same strain of bacteria avoids possible effects of mutants other than those intended, so an observed correlation would not necessarily be a response to the intended change. Our experiments demonstrate this situation for the correlation of microscopic bacterial motion and macroscopic colony growth. Both increase with the nutrient concentration in the lower concentration range, suggesting that the microscopic motion drives the growth of the colony. Microscopic inspection of the growth front supports this idea as bacteria in the growth region show collective motion forming whirls and jets near the growing tip of the colony. However, the speed of the bacteria and the speed of the colony growth level off at different nutrient concentrations and, therefore, the microscopic motion of the bacteria cannot be the origin of the colony growth. Experiments at different agar concentrations illustrate two extreme situations: for hard agar and high nutrient concentration, bacterial speed increases with increasing nutrient concentration, while the tip velocity remains essentially the same. In contrast, for soft agar and high nutrient concentration the bacteria reach their maximal speed, while the colony continues to grow faster when the nutrient concentration increases. There is still the possibility that the collective motion of the bacteria determines the colony growth instead of their average speed. Pushing the interface forward may require the concerted effort of a group of bacteria such as the observed jets and whirls. However, we have found that characteristic parameters of collective motion, such as the whirl correlation length or time, level off at different nutrient concentrations and, therefore, cannot be the driving force for the colonial growth. Thus we can conclude for P. dendritiformis (type T; tip-splitting) that the speed and pattern of bacterial motion and the overall colonial growth velocity are two largely independent parameters. At low nutrient concentration the bacterial speed and the colonial growth velocity both increase with increasing nutrient concentration, but at high nutrient concentrations the bacterial speed and colonial growth velocity are limited by different factors, which depend on the agar concentration. Downloaded from on March, by guest

11 If the bacterial motion is not the driving force for colonial growth, then what else determines it? The sharp interface is a result of the surface tension of the media which can be pushed forward either by a pressure generated within the colony or by reducing the surface tension. Our last experiment suggests strongly that the surfactant production determines colonial growth in P. dendritiformis (type T; tip-splitting), as increasing surfactant concentration increases the colonial growth speed but has little effect on bacterial motion. In summary, our results suggest that the microscopic bacterial motion, i.e. average speed as well as collective motion in whirls and jets, fulfills other functions for the colony than its bare overall growth. It is possible that our finding applies also to other bacteria that grow on surfaces; however, the generality of our finding remains to be determined since the relation between microscopic motions and colonial growth has not been systematically studied for a single strain of any other bacteria. ACKNOWLEDGMENTS We thank Eshel Ben-Jacob and Inna Brainis for providing the bacterial strain and the growth protocol. We are grateful to Rasika M. Harshey, George A. O Toole and Daniel B. Kearns for fruitful discussions. E.L.F. acknowledges support by the Robert A. Welch Foundation, and H.L.S. acknowledges support by the Sid W. Richardson Foundation. Downloaded from on March, by guest

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16 Figure Captions Fig.. P. dendritiformis bacterial colony grown on a.% (wt/vol) agar gel with g/l peptone. (A) h after inoculation. The inset in the lower right corner shows a close-up of the small region of the colony marked with an arrow; bar is mm. (B) Magnification of the region marked with the arrow in (A), showing three welldefined regions: Region III, where the bacteria at the outer edge of the colony are found in three layers and are very active; Region II, where the bacteria are in two layers and less active; and Region I, where the bacteria occupy a single layer and show little or no movement. The inset in the lower left corner shows a higher magnification of region I, where individual bacteria can be resolved. (C) Colony growth occurs only near the tips (the colony s edge), as illustrated in this figure obtained by subtracting the image at h from the image at h. (D) The growth velocity (. mm/h =. m/s) is given by the slope of this plot of the position x of the farthest edge of the colony as a function of time. The error bars indicate the standard deviation in measurements for colonies. Fig.. Microscopic speed measurements of a P. dendritiformis bacterial colony grown on a.% (wt/vol) agar gel with g/l peptone nutrient. (A) A phase contrast microscopic image of region III near the colony s edge, which is indicated by the dashed line. Arrows indicate the local displacement in a time interval of. s. Note the vortices and the jets between them. (B) An enlarged image of the vortex in the box in (A). (C) Vorticity field corresponding to the velocity field shown in (A). Deep red (counter-clockwise rotation) and blue (clockwise) regions correspond to intense vortices. (D) Average bacterial speed as a function of distance from the colony s edge, which is at position. Bacteria in each region have a distinct fairly uniform mean speed. Mean speed is calculated by averaging the speed vs. distance along many lines parallel to the horizontal direction in (A) for about frames, in different colonies. Standard deviation of experiments is too small to be visualized on this graph. Downloaded from on March, by guest

17 Fig.. Dependence of colony growth rate on nutrient concentration and on average bacterial speed. (A) Velocity of the tip of a growing colony as a function of peptone level for two agar concentrations. Each point corresponds to a constant velocity derived from the slope of the accumulated distance covered by the colony as a function of time, as in Fig. D. For both agar concentrations, increasing the peptone level initially increases the tip velocity. However, at some peptone level, the velocity value saturates, indicating an additional bottleneck. The food-limited region extends further for the lower agar concentration. (B) Tip velocity as a function of microscopic bacterial speed (in Region III; see Fig. B) for increasing peptone level (given in g/l for each point). For.% (wt/vol) agar concentration, there is a region where additional food significantly increases microscopic bacterial speed (nutrient-limited regime), but the tip velocity remains nearly the same. For the.% (wt/vol) agar, there is a region where additional food scarcely changes the microscopic bacterial speed (space-limited regime), yet the tip velocity increases dramatically. The error bars, in some cases smaller than the dots, indicate the standard deviation of three experiments. Fig.. Dependence of colony growth rate and bacterial speed on agar concentration. (A) Tip velocity as a function of agar concentration for peptone levels, and g/l. Each point was obtained from the slope of plots of x vs. t, as in Fig. D. Colonies grow faster for agar concentrations around.% (wt/vol), and more slowly for hard (>.% agar) or soft (<.%) gels. (B) Microscopic bacterial speed as a function of agar concentration for peptone levels of and g/l. The maximum mean speed of the bacteria is approximately twenty times as large as the colony growth velocity. The bacterial speed is greatest for agar concentrations around.% (wt/vol), and rapidly drops to zero at concentrations near.% (wt/vol). (C) Tip velocity as a function of microscopic bacterial speed for the indicated agar concentrations (in steps of.% (wt/vol)) for peptone levels of g/l (solid line) and g/l (dashed line). In each graph the error bars (in some cases smaller than the dot size) correspond to one standard deviation for three experiments. Downloaded from on March, by guest

18 Fig.. Whirl-correlation measurements in region III for a.% (wt/vol) agar gel containing g/l peptone. (A) Two-dimensional correlation map of V x (the velocity component along the propagation direction of the colony s tip), with the color bar indicating strong correlation for dark red to anti-correlation for dark blue. Note the two nodes of anti-correlation above and below the center, which indicate the presence of vortices. (B) A vertical slice through the center of (A). The dashed horizontal line crosses the curve at correlation=.; the segment on the horizontal axis (here y), denoted by a double-sided arrow, determines the whirlcorrelation length, about m in this case, as can be also seen in Fig. A. Fig.. Whirl-correlation length measurements of bacteria grown for various agar concentrations, as indicated in the graphs, for g/l peptone nutrient. (A) The whirl-correlation length increases monotonically with the microscopic bacterial speed. (B) The whirl-correlation length variation with the tip velocity; the curve is similar to Fig. C. In both (A) and (B) standard deviation of three experiments is too small to be visualized on this graph. Fig.. Colony growth rate and bacterial speed dependence on surfactant concentration. Colonies grown for h (A) without added surfactant and (B) with surfactant (.% (wt/vol) Brij) added in the growth media (.% (wt/vol) agar gels with g/l peptone nutrient). (C) Tip velocity and microscopic bacterial speed (each relative to the values with no added surfactant), as a function of surfactant concentration. Surfactant strongly affects the tip velocity but has little effect on the microscopic bacterial speed. The error bars correspond to the difference between experiments. Movie. Real-time microscopic motion of P. dendritiformis bacteria grown on a.% (wt/vol) agar gel with g/l peptone. The average bacterial speed is about. m/s. The movie was taken using phase contrast microscopy and shows Downloaded from on March, by guest

19 A B µm I II III C mm mm D x (mm) µm Time (h) Downloaded from on March, by guest

20 A B µm/s µm C rad/s. -. Bacterial speed (µm/s) D.. edge III II I - - distance from edge (µm) Downloaded from on March, by guest

21 . A.% agar Tip velocity (µm/s) Tip velocity (µm/s)...% agar Peptone (g/l). B...% agar Bacterial speed (µm/s) g/l peptone.% agar Downloaded from on March, by guest

22 Tip velocity (µm/s).... A g/l peptone g/l g/l Bacterial speed (µm/s).. B g/l peptone g/l Agar (%) Agar (%) Bacterial speed (µm/s) Tip velocity (µm/s).... C.% agar Downloaded from on March, by guest

23 Correlation Downloaded from on March, by guest

24 Whirl-correlation length (µm) Whirl-correlation length (µm) A.%.%.%.%.%.%.% agar... B.%.% Bacterial speed (µm/s).%.%.%.%.% agar... Tip velocity (µm/s) Downloaded from on March, by guest

25 A B C C Normalized speeds Tip velocity mm Bacterial speed... Brij concentration (%) Downloaded from on March, by guest