^{1}

^{2}

^{*}

^{2}

^{1}

The authors have declared that no competing interests exist.

Conceived and designed the experiments: JGM DGB KMW. Performed the experiments: KMW. Analyzed the data: JGM DGB KMW. Contributed reagents/materials/analysis tools: JGM DGB KMW. Wrote the paper: JGM DGB KMW.

The coral pathogen,

Bacteria use chemotaxis and motility search patterns to position themselves in chemical gradients, locate nutrient sources and initiate pathogenesis

Pathogenic and symbiotic

During coral infection,

The single, polar flagellated

A volume of 50 µL of cell suspension was added to an observation chamber constructed from two glass coverslips on a glass slide and covered with a third coverslip (^{−1} using a time-lapse module (Carl Zeiss AxioVision 4). Search strategies of

(a) Microscope slide, (b) cover slips, (c) center recordings, (d) edge recordings. A 3D numerical model depicts how oxygen diffuses from the top and the bottom of the coverslip labelled (e). Red depicts high oxygen concentrations with a vertical length of 5250 µm, whilst the blue depicts low oxygen concentrations. The chamber has a vertical length of 26,250 µm. The red dashed line illustrates the chamber cover, which sits on top of two cover slips.

A volume of 50 µL of 1/10 dilution of overnight culture was added to the created observation chamber. Triplicate transects were collected ranging from the oxic to the anoxic center of the observation chamber. Each transect consisted of 40 square eyepiece quadrats of 172 by 172 µm placed next to each other. The transect line spanned from the oxic to anoxic regions across 6,880 µm. At each quadrat a 10 second, 49-frame video was recorded. All distinguishable cells in each eyepiece quadrant video were tracked and analyzed. Cells were considered distinguishable if they remained in focus for at least 5 frames. All distinguishable cell trajectories were classified into one of three search patterns: run and reverse, straight swimming or 3-step flick (

Cell cultures and the headspaces were bubbled with either pure air or nitrogen for 10 minutes, and grown in conditions as mentioned above. Methylene blue and resazurin indicators (Sigma-Aldrich) were used to confirm whether a solution was saturated throughout the experiment

Microscope videos in oxic and anoxic conditions were collected at 5 minute intervals between 0 and 25 mins to measure the behavioral changes of

Numerical simulations of oxygen diffusion in the presence of bacteria were produced using the MatLab-derived COMSOL Multiphysics with the shown geometries using transport of dilute species with convection and diffusion models (^{3}), D is the diffusion coefficient (m^{2}/s), ^{3}s)) and in this case is used to signify flux,

The search pattern path starts at X; (a) cyclic 3-step flick search pattern, shown as a forward run, a reverse; seen as a 180° reorientation, and a 90° flick of the flagellum and repeat; (b) run and reverse and swimming search pattern. Run and reverse search patterns are characteristic of a 180° reorientation and reversal. Straight swimming search patterns shows no reversals; (c) 3-step flick search pattern trajectories (closed squares) collected from

^{−7} m.

All cell trajectories were classified into search patterns and the results were analyzed using SPSS Statistics (version 19.0). Independent sample t-tests were utilized to calculate P values to establish whether flick or non-flick search patterns differ in total numbers or speed across the two observation chamber locations. T-tests were also used to determine any statistical differences in search pattern behavior during the time series.

^{−1} (95% Confidence Interval (CI) = 2.0, n = 53), 21 µm s^{−1} (95% CI = 4.1, n = 29) and 21 µm s^{−1} (95% CI = 8.4, n = 13) respectively, were observed (^{−1} (95% CI = 2.6, n = 38), 20 µm s^{−1} (95% CI = 2.5, n = 46) and 18 µm s^{−1} (95% CI = 5.5, n = 14) respectively, were observed (

Search pattern | Observation chamber region | Aerobically growncultures | Anaerobicallygrown cultures | |||

Edge (Oxic) | Center (Anoxic) | |||||

Chamber | Surface | Chamber | Surface | |||

Straight swimming | 56 |
18 |
46 |
21 |
47 |
65 |

Run-reverse | 47 |
35 |
49 |
29 |
76 |
59 |

Combined non-flicks | 57 |
35 |
49 |
29 |
76 |
61 |

3-step flicks | 37 |
41 |
43 |
25 |
79 |
56 |

= maximum search pattern speed.

The chemotactic search pattern behavior of

(a) The mean total number of search patterns seen along the transect line consisting of 40 quadrat boxes (error bars = 95% CI); (b) Mean velocity (µm s^{−1}) of search patterns across the transect line (error bars = 95% CI). The location of the grey gradient lines represents the location where the oxygen transition occurs in Fig. 1 (orange and green area).

Search pattern velocities were shown to decrease 2-fold from oxic to anoxic environments (P = 0.002), from 63.2 µm s^{−1} (95% CI = 2.0, n = 58) to 21.9 µm s^{−1} (95% CI = 3.4, n = 32) respectively, although a large variation in search pattern velocity was identified (^{−1} from the 2000 µm distance location onwards (P = 0.578, 95% CI = 0.6, n = 400). Velocities did not differ significantly between the three types of search patterns identified, namely the flick, run and reverse and straight swimming (

Motile cells of ^{−1} (95% CI = 5.6, n = 38), 45 µm s^{−1} (95% CI = 8.4, n = 13) and 32 µm s^{−1} (95% CI = 0.7, n = 9) respectively, were observed (^{−1} (95% CI = 7.0, n = 5), 48 µm s^{−1} (95% CI = 6.9, n = 6) and 46 µm s^{−1} (95% CI = 3.5, n = 40) respectively (

During a 25-minute time series experiment, the search pattern numbers in anoxic conditions leveled off by the 5-minute mark, whereas in oxic conditions, search pattern numbers began to level off from 10 minutes onwards. In anoxic conditions, flick search patterns decreased by 14 times (P = 0.022, 95% CI = 8.0, n = 38), whilst non-flick patterns increased by 8 (95% CI = 3.4, n = 26) over the 25-minute experiment, though this result was not significant (P = 0.230) (

(a) The mean total number of search patterns seen at the edge of the cover slip and center of the cover slip over a 25 minute time series. The solid squares indicate the number of non-flick search patterns at an anoxic area of the chamber, open squares indicate the number of non-flick search patterns at an oxic area of the chamber, closed circles indicate the number of flicks search patterns at an anoxic area of the chamber, open circles indicate the number of flick search patterns at an oxic area of the chamber; (b) The mean search pattern velocities seen at oxic and anoxic regions of the cover slip chamber over a 25 minute time experiment. * = only 1 search pattern observed so no 95% CI value could be calculated; ? = missing data as no search patterns were identified.

Oxygen concentrations can differ between the surrounding water layer and the various microbiomes present in coral organisms, namely the surface mucus layer and coral tissue

Our results indicate that

Changes in search pattern behavior of

Blue dots represent bacteria that absorb oxygen as it diffuses past. The columns are 100 µm high (a and b). The relative concentration difference between blue and red is 30 mM. Orthogonalized spacings represent average cell distributions seen in the experiments with (a) for low cell density regions and (b) for cell cluster regions. Real distributions will be heterogenously rather than uniformly distributed. The diagonal in (c) is the rotated volume showing the z axis and the position of the bacteria in that direction.

A sample video recording of

(MOV)

Experimental number of recordings (n) for

(DOCX)

Experimental number of recordings (n) for

(DOCX)