DOI: 10.5061/dryad.tmpg4f51d
title: Short-Range C-Signaling Restricts Cheating Behavior during Myxococcus xanthus

description: Myxococcus xanthus uses short-range C-signaling to coordinate
 multicellular mound formation with sporulation during fruiting body
 development.  A csgA mutant deficient in C-signaling can cheat on
 wild type (WT) in mixtures and form spores disproportionately, but our
 understanding of cheating behavior is incomplete.  We subjected
 mixtures of WT and csgA cells at different ratios to co-development and
 used confocal microscopy and image analysis to quantify the arrangement
 and morphology of cells.  At a ratio of one WT to four csgA cells
 (1:4), mounds failed to form. At 1:2, only a few mounds and spores
 formed.  At 1:1, mounds formed with a similar number and
 arrangement of WT and csgA rods early in development, but later the number
 of csgA spores near the bottom of these nascent fruiting bodies (NFBs)
 exceeded that of WT.  This cheating after mound formation
 involved csgA forming spores at a greater rate while WT disappeared at a
 greater rate, either lysing or exiting NFBs.  At 2:1 and 4:1,
 csgA rods were more abundant than expected throughout the biofilm both
 before and during mound formation, and cheating continued after mound
 formation.  We conclude that C-signaling restricts cheating
 behavior by requiring sufficient WT cells in mixtures.  Excess
 cheaters may interfere with positive feedback loops that depend on the
 cellular arrangement to enhance C-signaling during mound building.
  Since long-range signaling could not likewise communicate the
 cellular arrangement, we propose that C-signaling was favored
 evolutionarily and that other short-range signaling mechanisms provided
 selective advantages in bacterial biofilm and multicellular animal
 development.
description: Images of nascent fruiting bodies were acquired with a Nikon A1
 Laser Scanning Confocal Microscope, which was configured on a Nikon Ti
 inverted platform with an XY automated stage and a 100X objective.
 Fluorescence from tdTomato were examined using a 560-nm laser for
 excitation and a 595/50 band pass emission filter. Fluorescence from
 mNeonGreen was examined using a 488 nm laser for excitation and a 525/50
 band pass emission filter. Images near the bottom of NFBs were the first
 optical section above the bottom of the well, in which cells could be
 clearly visualized (0.25 to 0.5 μm above the bottom of the
 well).
description: # Short-Range C-Signaling Restricts Cheating Behavior during *Myxococcus
 xanthus Development*
 [https://doi.org/10.5061/dryad.tmpg4f51d](https://doi.org/10.5061/dryad.tmpg4f51d) ## Description of the data and file structure \**Growth and development.  ***M. xanthus* was grown and submerged culture development.  Briefly, cells were grown at 32°C in CTTYE liquid medium (CTT with 0.2% yeast extract) with shaking at 350 rpm.  Development was performed in 8-well μ-slides (Ibidi #80826) with starvation buffer MC7 (10 mM morpholinepropanesulfonic acid [MOPS, pH 7.0], 1 mM CaCl2).  Cells from log-phase CTTYE cultures were collected by centrifugation and resuspended to 1,000 Klett units in MC7 containing 0.5 mM vanillate.  The cell suspensions were mixed as described in the text and the figure legends.  Each cell suspension (30 μL) was then added to 201 μL of MC7 containing 0.5 mM vanillate, and 200 μL was pipeted into each μ-slide well.  The μ-slide was wrapped in clear plastic and incubated at 32°C in a humid chamber.  **Strains.** | ***M. xanthus***\*\* strains\*\* | **Description** | | :------------------------------- | :----------------------------------------------- | | DK1622 | Laboratory wild-type strain (unlabeled) | | MRR33 | *csgA*::pRR028 (unlabeled) | | YH7 | DK1622 with MXAN\_0018-MXAN\_0019::pYH7  (green) | | YH11 | MRR33 with MXAN\_0018-MXAN\_0019::pYH8 (red) | **Experimental design. For all figures and analyses (except Figure 5 and Figure S12**) in the manuscript, half of the WT cells were labeled with the strain YH7 (green fluorescence), while the other half were unlabeled and represented by the strain DK1622. Similarly, for the *csgA* cells, half were labeled with the strain YH11 (red fluorescence), and the other half were unlabeled and represented by the strain MRR33. The z-stacks are listed in the table below. | **ID** | **Ratio** | **Hours post-starvation** | | :----- | :-------- | :------------------------- | | 1 | 4:1 | Indicated in the file name | | 2 | 4:1 | Indicated in the file name | | 3 | 4:1 | Indicated in the file name | | 4 | 4:1 | Indicated in the file name | | 5 | 4:1 | Indicated in the file name | | 6 | 2:1 | Indicated in the file name | | 7 | 2:1 | Indicated in the file name | | 8 | 2:1 | Indicated in the file name | | 9 | 2:1 | Indicated in the file name | | 10 | 2:1 | Indicated in the file name | | 11 | 1:1 | Indicated in the file name | | 12 | 1:1 | Indicated in the file name | | 13 | 1:1 | Indicated in the file name | | 14 | 1:1 | Indicated in the file name | | 15 | 1:1 | Indicated in the file name | | 16 | 1:2 | Indicated in the file name | | 17 | 1:2 | Indicated in the file name | | 18 | 1:2 | Indicated in the file name | | 19 | 1:2 | Indicated in the file name | | 20 | 1:2 | Indicated in the file name | **For Figures 5 and S12** in the manuscript**,** one-third of the WT cells were labeled with the strain YH7 (green fluorescence), while the remaining two-thirds were unlabeled and represented by the strain DK1622. Similarly, for the csgA cells, one-third were labeled with the strain YH11 (red fluorescence), and the remaining two-thirds were unlabeled and represented by the strain MRR33. The z-stacks are listed in the table below. | **Number** | **Name** | **Note** | | :--------- | :------------------------- | :-------------- | | 1 | r1 1\_1 18h 100X | Biofilm, 18 h | | 2 | r1 1\_1 18h 100X\_2 | Biofilm, 18 h | | 3 | r1 1\_2 18h 100X | Biofilm, 18 h | | 4 | r1 1\_2 18h 100X\_2 | Biofilm, 18 h | | 5 | r1 2\_1 18h 100X | Biofilm, 18 h | | 6 | r1 2\_1 18h 100X\_2 | Biofilm, 18 h | | 7 | r1 4\_1 18h 100X | Biofilm, 18 h | | 8 | r1 4\_1 18h 100X\_2 | Biofilm, 18 h | | 9 | r2 1\_1 18h 100X | Biofilm, 18 h | | 10 | r2 1\_2 18h 100X | Biofilm, 18 h | | 11 | r2 2\_1 18h 100X | Biofilm, 18 h | | 12 | r2 4\_1 18h 100X | Biofilm, 18 h | | 13 | r3 1\_1 18h 100X | Biofilm, 18 h | | 14 | r3 1\_2 18h 100X | Biofilm, 18 h | | 15 | r3 2\_1 18h 100X | Biofilm, 18 h | | 16 | r3 4\_1 18h 100X | Biofilm, 18 h | | 17 | r4 1\_1 18h 100X | Biofilm, 18 h | | 18 | r4 1\_2 18h 100X | Biofilm, 18 h | | 19 | r4 2\_1 18h 100X | Biofilm, 18 h | | 20 | r4 4\_1 18h 100X | Biofilm, 18 h | | 21 | r5 1\_1 18h 100X | Biofilm, 18 h | | 22 | r5 1\_2 18h 100X | Biofilm, 18 h | | 23 | r5 2\_1 18h 100X | Biofilm, 18 h | | 24 | r5 4\_1 18h 100X | Biofilm, 18 h | | 25 | r6 1\_1 18h 100X | Biofilm, 18 h | | 26 | r6 1\_2 18h 100X | Biofilm, 18 h | | 27 | r6 2\_1 18h 100X | Biofilm, 18 h | | 28 | r6 4\_1 18h 100X | Biofilm, 18 h | | 29 | r1 1\_1 24h 100X NFB | NFB, 24 h | | 30 | r1 1\_2 24h 100X NFB | NFB, 24 h | | 31 | r1 1\_2 24h 100X NFB\_2 | NFB, 24 h | | 32 | r1 2\_1 24h 100X NFB | NFB, 24 h | | 33 | r1 4\_1 24h 100X NFB | NFB, 24 h | | 34 | r1 4\_1 24h 100X NFB\_2 | NFB, 24 h | | 35 | r2 1\_1 24h 100X NFB | NFB, 24 h | | 36 | r2 1\_2 24h 100X NFB | NFB, 24 h | | 37 | r2 2\_1 24h 100X NFB | NFB, 24 h | | 38 | r2 4\_1 24h 100X NFB | NFB, 24 h | | 39 | r3 1\_1 24h 100X NFB | NFB, 24 h | | 40 | r3 1\_2 24h 100X NFB | NFB, 24 h | | 41 | r3 2\_1 24h 100X NFB | NFB, 24 h | | 42 | r3 4\_1 24h 100X NFB | NFB, 24 h | | 43 | r4 1\_1 24h 100X NFB | NFB, 24 h | | 44 | r4 1\_2 24h 100X NFB | NFB, 24 h | | 45 | r4 1\_2 24h 100X NFB\_2 | NFB, 24 h | | 46 | r4 2\_1 24h 100X NFB | NFB, 24 h | | 47 | r4 4\_1 24h 100X NFB | NFB, 24 h | | 48 | r5 1\_1 24h 100X NFB | NFB, 24 h | | 49 | r5 1\_2 24h 100X NFB | NFB, 24 h | | 50 | r5 1\_2 24h 100X NFB\_2 | NFB, 24 h | | 51 | r5 2\_1 24h 100X NFB | NFB, 24 h | | 52 | r5 4\_1 24h 100X NFB | NFB, 24 h | | 53 | r1 1\_1 24 h 100X 1 fov | Proximal,  24 h | | 54 | r1 1\_2 24 h 100X 1 fov | Proximal,  24 h | | 55 | r1 2\_1 24 h 100X 1 fov | Proximal,  24 h | | 56 | r1 4\_1 24 h 100X 1 fov | Proximal,  24 h | | 57 | r2 1\_1 24 h 100X 1 fov | Proximal,  24 h | | 58 | r2 1\_2 24 h 100X 1 fov | Proximal,  24 h | | 59 | r2 2\_1 24 h 100X 1 fov | Proximal,  24 h | | 60 | r2 4\_1 24 h 100X 1 fov | Proximal,  24 h | | 61 | r3 1\_1 24 h 100X 1 fov | Proximal,  24 h | | 62 | r3 1\_2 24 h 100X 1 fov | Proximal,  24 h | | 63 | r3 2\_1 24 h 100X 1 fov | Proximal,  24 h | | 64 | r3 4\_1 24 h 100X 1 fov | Proximal,  24 h | | 65 | r4 1\_1 24 h 100X 1 fov | Proximal,  24 h | | 66 | r4 1\_2 24 h 100X 1 fov | Proximal,  24 h | | 67 | r4 1\_2 24 h 100X 1 fov\_2 | Proximal,  24 h | | 68 | r4 2\_1 24 h 100X 1 fov | Proximal,  24 h | | 69 | r4 4\_1 24 h 100X 1 fov | Proximal,  24 h | | 70 | r5 1\_1 24 h 100X 1 fov | Proximal,  24 h | | 71 | r5 1\_2 24 h 100X 1 fov | Proximal,  24 h | | 72 | r5 1\_2 24 h 100X 1 fov\_2 | Proximal,  24 h | | 73 | r5 2\_1 24 h 100X 1 fov | Proximal,  24 h | | 74 | r5 4\_1 24 h 100X 1 fov | Proximal,  24 h | | 75 | r1 1\_1 24 h 100X center | Distal, 24 h | | 76 | r1 1\_2 24 h 100X center | Distal, 24 h | | 77 | r1 2\_1 24 h 100X center | Distal, 24 h | | 78 | r1 4\_1 24 h 100X center | Distal, 24 h | | 79 | r2 1\_1 24 h 100X center | Distal, 24 h | | 80 | r2 1\_2 24 h 100X center | Distal, 24 h | | 81 | r2 2\_1 24 h 100X center | Distal, 24 h | | 82 | r2 4\_1 24 h 100X center | Distal, 24 h | | 83 | r3 1\_1 24 h 100X center | Distal, 24 h | | 84 | r3 1\_2 24 h 100X center | Distal, 24 h | | 85 | r3 2\_1 24 h 100X center | Distal, 24 h | | 86 | r3 4\_1 24 h 100X center | Distal, 24 h | | 87 | r4 1\_1 24 h 100X center | Distal, 24 h | | 88 | r4 1\_2 24 h 100X center | Distal, 24 h | | 89 | r4 2\_1 24 h 100X center | Distal, 24 h | | 90 | r4 4\_1 24 h 100X center | Distal, 24 h | | 91 | r5 1\_1 24 h 100X center | Distal, 24 h | | 92 | r5 1\_2 24 h 100X center | Distal, 24 h | | 93 | r5 2\_1 24 h 100X center | Distal, 24 h | | 94 | r5 4\_1 24 h 100X center | Distal, 24 h | | 95 | r1 1\_1 NFB1 | NFB, 30 h | | 96 | r1 1\_2 NFB1 | NFB, 30 h | | 97 | r1 2\_1 NFB1 | NFB, 30 h | | 98 | r1 4\_1 NFB1 | NFB, 30 h | | 99 | r2 1\_1 NFB1 | NFB, 30 h | | 100 | r2 1\_2 NFB1 | NFB, 30 h | | 101 | r2 2\_1 NFB1 | NFB, 30 h | | 102 | r2 4\_1 NFB1 | NFB, 30 h | | 103 | r3 1\_1 NFB1 | NFB, 30 h | | 104 | r3 1\_2 NFB1 | NFB, 30 h | | 105 | r3 2\_1 NFB1 | NFB, 30 h | | 106 | r3 4\_1 NFB1 | NFB, 30 h | | 107 | r4 1\_1 NFB1 | NFB, 30 h | | 108 | r4 1\_2 NFB1 | NFB, 30 h | | 109 | r4 2\_1 NFB1 | NFB, 30 h | | 110 | r4 4\_1 NFB1 | NFB, 30 h | | 111 | r5 1\_1 NFB1 | NFB, 30 h | | 112 | r5 1\_2 NFB1 | NFB, 30 h | | 113 | r5 2\_1 NFB1 | NFB, 30 h | | 114 | r5 4\_1 NFB1 | NFB, 30 h | | 115 | r6 1\_1 NFB1 | NFB, 30 h | | 116 | r6 1\_2 NFB1 | NFB, 30 h | | 117 | r6 2\_1 NFB1 | NFB, 30 h | | 118 | r6 4\_1 NFB1 | NFB, 30 h | | 119 | r1 1\_1 FOV1 | Proximal, 30 h | | 120 | r1 1\_2 FOV1 | Proximal, 30 h | | 121 | r1 2\_1 FOV1 | Proximal, 30 h | | 122 | r1 4\_1 FOV1 | Proximal, 30 h | | 123 | r2 1\_1 FOV1 | Proximal, 30 h | | 124 | r2 1\_2 FOV1 | Proximal, 30 h | | 125 | r2 2\_1 FOV1 | Proximal, 30 h | | 126 | r2 4\_1 FOV1 | Proximal, 30 h | | 127 | r3 1\_1 FOV1 | Proximal, 30 h | | 128 | r3 1\_2 FOV1 | Proximal, 30 h | | 129 | r3 2\_1 FOV1 | Proximal, 30 h | | 130 | r3 4\_1 FOV1 | Proximal, 30 h | | 131 | r4 1\_1 FOV1 | Proximal, 30 h | | 132 | r4 1\_2 FOV1 | Proximal, 30 h | | 133 | r4 2\_1 FOV1 | Proximal, 30 h | | 134 | r4 4\_1 FOV1 | Proximal, 30 h | | 135 | r4 4\_1 FOV2 | Proximal, 30 h | | 136 | r5 1\_1 FOV1 | Proximal, 30 h | | 137 | r5 1\_2 FOV1 | Proximal, 30 h | | 138 | r5 2\_1 FOV1 | Proximal, 30 h | | 139 | r5 4\_1 FOV1 | Proximal, 30 h | | 140 | r5 4\_1 FOV2 | Proximal, 30 h | | 141 | r6 1\_1 FOV1 | Proximal, 30 h | | 142 | r6 1\_2 FOV1 | Proximal, 30 h | | 143 | r6 2\_1 FOV1 | Proximal, 30 h | | 144 | r6 4\_1 FOV1 | Proximal, 30 h | | 145 | r6 4\_1 FOV2 | Proximal, 30 h | | 146 | r1 1\_1 center1 | Distal, 30 h | | 147 | r1 1\_2 center1 | Distal, 30 h | | 148 | r1 2\_1 center1 | Distal, 30 h | | 149 | r1 4\_1 center1 | Distal, 30 h | | 150 | r2 1\_1 center1 | Distal, 30 h | | 151 | r2 1\_2 center1 | Distal, 30 h | | 152 | r2 2\_1 center1 | Distal, 30 h | | 153 | r2 4\_1 center1 | Distal, 30 h | | 154 | r3 1\_1 center1 | Distal, 30 h | | 155 | r3 1\_2 center1 | Distal, 30 h | | 156 | r3 2\_1 center1 | Distal, 30 h | | 157 | r3 4\_1 center1 | Distal, 30 h | | 158 | r4 1\_1 center1 | Distal, 30 h | | 159 | r4 1\_2 center1 | Distal, 30 h | | 160 | r4 2\_1 center1 | Distal, 30 h | | 161 | r4 4\_1 center1 | Distal, 30 h | | 162 | r5 1\_1 center1 | Distal, 30 h | | 163 | r5 1\_2 center1 | Distal, 30 h | | 164 | r5 2\_1 center1 | Distal, 30 h | | 165 | r5 4\_1 center1 | Distal, 30 h | | 166 | r6 1\_1 center1 | Distal, 30 h | | 167 | r6 1\_2 center1 | Distal, 30 h | | 168 | r6 2\_1 center1 | Distal, 30 h | | 169 | r6 4\_1 center1 | Distal, 30 h | ## Code/software The original raw image data was in .nd2 file, which can be opened with ImageJ, a free open-source tool.