Physical factors contributing to regulation of bacterial surface motility 1
Ben Rhodeland
1
, Kentaro Hoeger
1
, and Tristan Ursell
1,2,3
2
3
Department of Physics
1
, Institute of Molecular Biology
2
, Materials Science Institute
3
4
University of Oregon, Eugene OR 97403 5
6
Microbes routinely face the challenge of acquiring territory and resources on wet surfaces. Cells move 7
in large groups inside thin, surface-bound water layers, often achieving speeds of 30 µm/s within this 8
environment, where viscous forces dominate over inertial forces (low Reynolds number). The canonical 9
Gram-positive bacterium Bacillus subtilis is a model organism for the study of collective migration over 10
surfaces with groups exhibiting motility on length scales three orders of magnitude larger than 11
themselves within a few doubling times. Genetic and chemical studies clearly show that the secretion 12
of endogenous surfactants and availability of free surface water are required for this fast group motility. 13
Here we show that: (i) water availability is a sensitive control parameter modulating an abiotic 14
jamming-like transition that determines whether the group remains fluidized and therefore collectively 15
motile, (ii) groups self-organize into discrete layers as they travel, (iii) group motility does not require 16
proliferation, rather groups are pulled from the front, and (iv) flow within expanding groups is capable 17
of moving material from the parent colony into the expanding tip of a cellular dendrite with implications 18
for expansion into regions of varying nutrient content. Together, these findings illuminate the physical 19
structure of surface-motile groups and demonstrate that physical properties, like cellular packing 20
fraction and flow, regulate motion from the scale of individual cells up to length scales of centimeters. 21
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Introduction 22
In their search for resources microbes contend with physically distinct environments ranging from 23
soft surfaces to bulk Newtonian fluids and complex fluids like mucus. In bulk fluid environments, canonical 24
microbes like Escherichia coli (1) and Bacillus subtilis (2, 3) ascend favorable chemical gradients via run-25
and-tumble chemotaxis (4). This mechanism of gradient ascent requires both flagellar-mediated motility 26
and a complex system of phosphorylation-memory and chemical sensors on the bacterial surface, that 27
together regulate the run-tumble transition frequency (5). In contrast, bacterial surface motility has 28
different requirements and inputs, and different species have evolved distinct modalities of surface 29
motion. For instance, in the predatory species Myxococcus xanthus, individual cells move in back-and-30
forth motions that employ two distinct sets of protein machinery for ‘twitching’ and ‘gliding’ motility, and 31
cells assemble into larger motile groups that traverse surfaces as monolayers (6, 7). Many other species, 32
including the opportunistic pathogens Serratia marcescens (8, 9) and Proteus mirabilis (10, 11), also form 33
large groups of motile cells that are capable of rapidly expanding over surfaces, in some cases even against 34
bulk fluid flow (12). Similarly, when present in sufficient numbers Paenibacillus dendritiformis form 35
intricate fractal-like patterns on soft agar surfaces in response to lateral chemical gradients (13, 14). Even 36
baker’s yeast have been observed to exhibit group movement over fluid surfaces (15). Other species 37
exhibit surface motility in response to non-chemical fields; for instance the cyanobacterium Synechocystis 38
is phototactic, responding to incident light by asymmetrically extending and retracting pili from its surface 39
to create a biased random walk toward a light source (16, 17). Crucial to its motion, Synechocystis modifies 40
the local surface environment by secreting exopolysaccharides, and only when enough cells have 41
participated in such surface modification can the group move toward the light source. These examples 42
demonstrate that in response to various gradients, microbes have evolved distinct sensing capabilities 43
and modalities of motion to acquire resources and respond to selective pressures on surfaces. Despite 44
their differences, surface motility in all of these species (and even in abiotic systems (18)) appears to be a 45
collective phenomenon, requiring the motion of and/or surface modification by large numbers of cells 46
(19, 20). Uncovering biochemical and genetic factors that regulate motion is crucial for understanding 47
how each species executes surface motility, but those factors are only part of the full picture. Physical 48
forces that produce, regulate, and guide microbial group motion on surfaces may be relevant across 49
species and contexts, and are thus integral to our understanding of microbial ecology in natural 50
environments and will expand the suite of design tools for engineering microbial systems. 51
Modeling plays an important role in these systems because it has the potential to connect 52
experimental observations with physical forces and regulators of motion (21–29). Different models posit 53
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qualitatively distinct physical mechanisms for group motility, with basal assumptions motivated by 54
species-specific attributes. In P. dendritiformis growth and spreading of cells in dendritic patterns are 55
modeled as a diffusion-limited conversion of nutrients into biomass at the growing tips of the dendrite 56
(13, 30). Cellular motion is thought to rely on chemotaxis that follows nutrient gradients that are created 57
by metabolic consumption of the colonies themselves (22, 30, 31). Together these effects recapitulate 58
many of the classes of bacterial surface patterning observed in experiments (26, 32–36). However, within 59
a dense and active (e.g. swarming) group whose velocity correlations rapidly decay on the length scale of 60
a few cells (21), it is unclear how individuals could effectively modulate their tumble frequency and 61
accumulate sufficiently persistent runs to deliberately bias their random walks and hence execute run-62
and-tumble chemotaxis. Indeed, previous work in B. subtilis (37–40) (and Pseudomonas aeruginosa (41), 63
Salmonella enterica (42), and Escherichia coli (43)) shows that neither chemotaxis nor motility of 64
individuals are necessary for rapid and outward-directed surface motility. Other models of surface motility 65
(e.g. swarming or sliding) in B. subtilis or P. aeruginosa focus on the role of surfactants (23, 44) and/or 66
osmotic potential (24), both of which appear to be crucial for rapid surface motility in those species (37, 67
45–47). 68
While cell density is clearly an important factor in motion, in so much as many cells are required 69
for collective movement, and genetics implicates endogenous surfactant production as a physical 70
requirement for collective motion, it remains unclear how density, material flow, and dendrite structure 71
contribute to regulation of this ubiquitous behavior. In this work we combined high resolution video and 72
time-lapse microscopy with computational image processing to clarify the relative contributions of 73
density, flow and structure by examining the surface motility of the extensively-studied Gram-positive 74
bacterium B. subitilis (5, 24, 48, 49). Within a few hours of deposition, a small central inoculum of wild-75
type cells can rapidly colonize the entire surface of a wet 10 cm agar plate via apparent swarming motility 76
(50). Such group motility over soft surfaces has been shown to depend on the secretion of ‘surfactin’, a 77
bacterially produced surfactant and wetting agent (22, 23, 25, 37, 51–53). Functional knockouts for 78
surfactin production (Δsrf) result in a phenotype where individual cells are still motile and chemotactic in 79
bulk fluid, but bacterial groups cannot move across surfaces (37, 40, 54). Localized secretion of surfactin 80
is thought to generate a gradient in surface tension, and thus produce collective motion via the Marangoni 81
force (23, 55, 56). 82
Building on that biophysical picture, we found evidence that the movement of B. subtilis over soft 83
surfaces was regulated by cell density, whereby groups of cells are subject to a jamming-fluidization 84
transition (57) that correlates with packing fraction (and thus water availability). These results 85
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complement findings that the viscosity of bacterial suspensions depends on cell concentration, shear rate, 86
and level of motility (58, 59). Further, we show that such groups operate in two distinct modalities of 87
motion: groups can move without growth as ‘islands’ that translocate independent of the parent colony, 88
or dendrites can extend with large-scale flow of material from the parent colony to the dendrite tip. We 89
used a chemotropic assay to show that when connected to a nutrient-rich parent colony, extending 90
dendrites can venture into nutrient-barren regions, while colonies that originate in nutrient-barren 91
regions are not collectively motile. These data suggest that, in addition to genetically regulated production 92
of surfactants, multiple physical and abiotic factors regulate motion independent of the chemotactic or 93
motile abilities of the constituent cells, and that groups move differently when presented with anisotropic 94
nutrient environments. When combined with previous work, these data suggest a model in which 95
individual motility and subsequent swarming are mechanisms that maintain a fluidized state on which 96
surface-tension gradient forces (Marangoni forces) and osmotically driven colony hydration can act to 97
precipitate group motion over a surface, and that a jamming-like transition, like those found in other 98
macroscopic granular systems (57, 60–65), may be a key regulator of whether bacterial groups are able 99
to move. 100
101
Results 102
Bacillus subtilis is a model motile Gram-positive bacterium, capable of sensing and responding to 103
its chemical environment in bulk fluid via run-and-tumble chemotaxis (2, 5, 66). On wet surfaces wild-type 104
B. subtilis rapidly move out from a central inoculum (67), apparently via collective swarming motility (50) 105
that requires secretion of the endogenous bio-surfactant ‘surfactin’. Mutants that lack the ability to 106
produce surfactin (∆srf) do not exhibit collective motility from their central inoculum (37) (and Movie S1). 107
We inoculated small, dense (OD ~ 10 - 20) droplets of wild-type B. subtilis on soft (~0.5% w/v agarose) 108
nutrient-rich surfaces. After a brief quiescent phase, groups of cells rapidly expanded over the surface in 109
a dendritic pattern that reached the edge of the plate (~5 cm of travel) in less than 6 hours (Movie S2). 110
Dendrites robustly moved outward away from the original point of inoculation into fresh territory at an 111
average group motility rate of ~5 µm/s and up to ~15 µm/s (SI Fig. 1). Cells within the dendrites were 112
highly motile, exhibiting swarming motility (37) with individual cells moving at rates up to ~30 µm/s in 113
highly circuitous paths. Consistent with previous work (37, 38), we confirmed that mutants lacking the 114
ability to run (tumble-only, ∆CheY), lacking the ability to tumble (run-only, ∆CheB), and mutants lacking 115
flagella (∆hag) were all able to exhibit rapid surface motility, albeit with some differences in dendrite 116
speed and spreading pattern (SI Fig. 2, Movies S3 - S5). 117
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In our early observations we noted transitions in the motile state of groups, wherein cells within 118
a contiguous region appeared to be either highly motile and moving as a group or immobilized on both 119
the individual and group levels. These distinct states of motion were frequently observed at the same 120
time, and cellular groups were observed transitioning between these states on the time scale of a minute, 121
too short to be accounted for by phenotypic changes. 122
123
Movement is regulated by a jamming-like transition 124
While surface motility of B. subtilis is robust to genetic manipulations of chemotaxis and motility, 125
it was very sensitive to gel stiffness as measured by agarose concentration (37). Below ~0.4% agarose by 126
weight the gel is sufficiently porous that bacteria can penetrate and swim within it, akin to canonical swim 127
plates (50, 68). Above ~0.7% agarose, limited water availability hinders surface motility (50), leading to 128
expansion across plates through the replication of sessile cells at the leading edge of growth, as seen in P. 129
dentritiformis (dendrite-like) or E. coli (growing circular colonies), both of which expand at much slower 130
rates (31, 69, 70). Thus, across a relatively narrow range of agarose percentages, groups of B. subtilis 131
exhibit three qualitatively distinct behaviors: swimming through agarose (below ~0.4%), rapid surface 132
motility (between ~0.4% and ~0.7%), and slow growth by replication (above ~0.7%). Why does the 133
transition from rapid surface motility to slow proliferative growth occur over such a narrow range of 134
agarose stiffness, and correspondingly, water availability? 135
Many previous studies of B. subtilis characterized spreading and attendant morphological 136
behaviors at the length-scale of colonies (mm to cm) and on the timescale of bacterial replication, imaging 137
colony morphology and spreading with minutes or hours between frames (26, 32, 37, 50). These excellent 138
studies revealed much of what is known about spreading phenotypes, their genetic mechanisms, and 139
biochemical correlates. We performed high temporal and spatial resolution imaging of spreading colonies 140
to illuminate processes that potentiate spreading. We captured images at 30 or 60 frames per second 141
with spatial resolution of 5 µm/pixel or 15 µm/pixel, respectively, both of which enabled us to see intensity 142
variations produced by the movements of cells within the swarm. We wrote a custom image analysis script 143
that measured a scalar correlate of motion as a function of position through time. Briefly, the algorithm 144
measures the mean absolute value of local intensity fluctuations at a position across a set of N (usually 5 145
– 7) frames and thus reports on the level of motile activity at each position through time (see Methods). 146
With this ‘activity’ filter, we were able to visualize on the scale of 10s to 100s of microns which parts of 147
the colony were actively motile and in which parts cells were stationary (Fig. 1A). 148
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