Cell density, alignment, and orientation correlate with C-signal–dependent gene expression during Myxococcus xanthus development

Abstract

Starving Myxococcus xanthus bacteria use short-range C-signaling to coordinate their movements and construct multicellular mounds, which mature into fruiting bodies as rods differentiate into spherical spores. Differentiation requires efficient C-signaling to drive the expression of developmental genes, but how the arrangement of cells within nascent fruiting bodies (NFBs) affects C-signaling is not fully understood. Here, we used confocal microscopy and cell segmentation to visualize and quantify the arrangement, morphology, and gene expression of cells near the bottom of NFBs at much higher resolution than previously achieved. We discovered that “transitioning cells” (TCs), intermediate in morphology between rods and spores, comprised 10 to 15% of the total population. Spores appeared midway between the center and the edge of NFBs early in their development and near the center as maturation progressed. The developmental pattern, as well as C-signal–dependent gene expression in TCs and spores, were correlated with cell density, the alignment of neighboring rods, and the tangential orientation of rods early in the development of NFBs. These dynamic radial patterns support a model in which the arrangement of cells within the NFBs affects C-signaling efficiency to regulate precisely the expression of developmental genes and cellular differentiation in space and time. Developmental patterns in other bacterial biofilms may likewise rely on short-range signaling to communicate multiple aspects of cellular arrangement, analogous to juxtacrine and paracrine signaling during animal development.

Regulation of late-acting operons by three transcription factors and a CRISPR-Cas component duringMyxococcus xanthusdevelopment

“…Thus, TCs comprised 15% of the total population at 24 h, before sonication-resistant spores were observed (Fig. 3), in agreement with in situ observations using confocal microscopy (46). The fadI mutant showed about half as many TCs as the WT strain at most time points (Fig.…”

“…We used the fixed samples to quantify “sonication-sensitive cells” microscopically (i.e., the total number of cells observed in the fixed sample minus the number of sonication-resistant spores observed in the corresponding untreated sample). The vast majority of sonication-sensitive cells were rod-shaped, but we also observed a small percentage of “transitioning cells” (TCs) intermediate in morphology between rods and spores (46) in some of the fixed samples.…”

“…Building on the recent observation of TCs in nascent fruiting bodies (46), we devised a method that allowed us to visualize and enumerate TCs in samples of the starved mutants (Fig. S1B).…”

“…The levels of MrpC, FruA, and CsgA normally rise in starving rods engaged in mound building (3), but the addition of sufficient nutrients triggers rapid proteolysis of MrpC and prevents the normal increase in transcripts for proteins involved in sporulation (38,39). Combinatorial control of gene expression by MrpC and FruA appears to ensure that only starving rods (in which MrpC and FruA are synthesized and stably accumulate) aligned in mounds (which enhances shortrange C-signaling between rods, presumably activating FruA within them) commit to spore formation (7,19,31,38,46). Among the genes combinatorially controlled by MrpC and FruA, early studies showed that mutations in the dev operon can cause severe defects in sporulation (47)(48)(49)(50).…”

Multiple roads lead to Rome: unique morphology and chemistry of endospores, exospores, myxospores, cysts and akinetes in bacteria

“…Myxospores are not as resistant as endospores, but they show higher resistance to high temperature, desiccation, UV and sonication than the corresponding vegetative cells [24]. This unique multicellular strategy to form spores has been studied more than encystment, but many genetic markers and mechanisms that lead to the production of myxospores are still unknown [25][26][27].…”