In situ quantitation of the spatial scale of calling distances and population density-independent N-acylhomoserine lactone-mediated communication by rhizobacteria colonized on plant roots

Abstract

We used computer-assisted microscopy at single cell resolution to quantify the in situ spatial scale of N-acylhomoserine lactone (AHL)-mediated cell-to-cell communication of Pseudomonas putida colonized on tomato and wheat root surfaces. The results of this in situ quantification study on close-to-natural surfaces challenge the conventional view of a quorum group requirement of high cell densities for this type of bacterial communication. In situ image analysis indicated that the effective 'calling distance' on root surfaces was most frequent at 4-5 microm, extended to 37 microm in the root tip/elongation zone and further out to 78 microm in the root hair zone. The spatial scale of these calling distances is very long-range in proportion to the size of individual bacteria. Geostatistical modeling analysis implicated the importance of AHL-gradients mediating effective communication between remote cells. We conclude that AHL-mediated cell-to-cell communication occurs not only within dense populations, but also in very small groups and over long ranges between individual bacteria, and therefore this cellular activity is more commonplace and effective than hitherto predicted. We propose that this cell-to-cell communication is governed more by the in situ spatial proximity of cells within AHL-gradients than the requirement for a quorum group of high population density.

Use of CMEIAS Image Analysis Software to Accurately Compute Attributes of Cell Size, Morphology, Spatial Aggregation and Color Segmentation that Signify in Situ Ecophysiological Adaptations in Microbial Biofilm Communities

“…This process occurs when external signal molecules accumulate sufficiently to affect neighboring cells [4,22,65]. Earlier studies [65] using laser scanning confocal fluorescence microscopy and CMEIAS image analysis indicated that two individual cells (one a producer and the other a sensor) are sufficient to fulfill the quorum requirement for N-acylhomoserine lactone (AHL)-negotiated communication while they colonize plant roots. The "calling distance" between pairs of communicating cells during colonization had a mode of 4-5 µm and extended out to a maximum of 78 μm [65] (equivalent to two people conversing while at opposite ends of a soccer field).…”

“…Earlier studies [65] using laser scanning confocal fluorescence microscopy and CMEIAS image analysis indicated that two individual cells (one a producer and the other a sensor) are sufficient to fulfill the quorum requirement for N-acylhomoserine lactone (AHL)-negotiated communication while they colonize plant roots. The "calling distance" between pairs of communicating cells during colonization had a mode of 4-5 µm and extended out to a maximum of 78 μm [65] (equivalent to two people conversing while at opposite ends of a soccer field). Further geospatial analysis indicated that this process of cell-cell communication is controlled more by cell location within diffusion gradients of the signal molecules made by their neighbors than by a quorum requirement of high population density [65].…”

“…The "calling distance" between pairs of communicating cells during colonization had a mode of 4-5 µm and extended out to a maximum of 78 μm [65] (equivalent to two people conversing while at opposite ends of a soccer field). Further geospatial analysis indicated that this process of cell-cell communication is controlled more by cell location within diffusion gradients of the signal molecules made by their neighbors than by a quorum requirement of high population density [65]. Thus, bacterial communication is more common than previously anticipated by the dogma that it only occurs at very high population densities [4,65].…”

“…These figures provide compelling evidence that the geospatially autocorrelated clustering behavior was stronger and more abundant when the microbes colonized the polystyrene substratum. An interesting application of color-discriminated microbial ecophysiology utilizes engineered strains to report cell-cell communication that triggers the expression of specific genes [4,65]. This process occurs when external signal molecules accumulate sufficiently to affect neighboring cells [4,22,65].…”

“…An interesting application of color-discriminated microbial ecophysiology utilizes engineered strains to report cell-cell communication that triggers the expression of specific genes [4,65]. This process occurs when external signal molecules accumulate sufficiently to affect neighboring cells [4,22,65]. Earlier studies [65] using laser scanning confocal fluorescence microscopy and CMEIAS image analysis indicated that two individual cells (one a producer and the other a sensor) are sufficient to fulfill the quorum requirement for N-acylhomoserine lactone (AHL)-negotiated communication while they colonize plant roots.…”