Spatial control of cell differentiation in <em>Myxococcus xanthus</em>

Abstract

How spatial patterns of differentiated cells arise is a central issue for animal and plant development. Myxococcus xanthus and other myxobacteria differentiate spores in response to nutrient deprivation. Although most bacteria sporulate individually, myxobacteria build large structured masses of spores, called fruiting bodies. Under nutrient-rich conditions, M. xanthus grows and divides as rod-shaped cells. When its development is induced by starvation, a hundred thousand cells contribute to building a fruiting body, whose shape is species-specific. Cells that have entered into the fruiting body finally differentiate into environmentally resistant myxospores, which can survive years without nutrients. However, not all of the starvation-induced cells become spores. Cells within fruiting bodies become spores, cells outside and between these multicellular structures remain rod-shaped and nonresistant (1). These cells, called peripheral rod cells, never become spores, despite their synthesis of two sporulation proteins, Tps and C (1). Dworkin and Gibson (2) showed that every cell innately has the capacity to become a spore. A difference in the developmental fate of peripheral rods and of fruiting body cells constitutes a spatial pattern that needs to be explained.

Most patterns involve cell-to-cell signaling, and sporulation depends on C-signaling. Ordinarily, each cell is simultaneously a transmitter and a receiver of the C-signal. The CsgA protein, on the surface of one cell, induces three processes in a contiguous cell (3–5). One consequence of signal reception is an enhancement of csgA expression. After initial episodes of C-signaling, an increase in the amount of CsgA protein per responding cell is observed (6, 7). Although the cell's receptor for C-signal has not yet been identified, it has been established that CsgA protein on one cell engenders an activating modification (probably phosphorylation) of the FruA response regulator protein in the other cell (8, 9). Activation of FruA signals the frz phospho-relay (10) to methylate FrzCD (11, 12), and to adjust the movement parameters of responding cells (13). Increased speed, increased movement intervals, and decreased stop frequency are the consequences, and these changes allow the cells to congregate into mound-shaped aggregates. Finally, activated FruA induces sporulation. However, it has been shown that sporulation requires a higher intensity of C-signaling than aggregation (6, 14). C-signal-dependent csgA expression has a lower threshold than aggregation or sporulation. As C-signaling intensity rises with csgA expression, it first reaches the threshold for aggregation, then, enhanced by positive feedback, it finally reaches the threshold for sporulation.

An operon was discovered several years ago that is expressed at high level, but only in a fraction of developing cells. This operon was originally identified in a search for developmentally regulated genes using the transposable element, Tn5lac (15). Tn5lac has a promoterless lacZ preceded by translation stops in all three reading frames (16). Expression in single cells was measured, using fluorescence activated cell sorting (FACS) to reveal the cell-to-cell distribution of β-galactosidase activity (17). Most developmentally regulated Tn5lac strains, like Ω4499 and Ω4506 (15), showed unimodal expression of β-galactosidase. Having been induced to develop for a given time, all their cells were expressing lacZ to similar levels distributed continuously about a single strain-characteristic modal value. But Tn5lac insertion Ω4473 showed bimodal expression. Some individual cells expressed the locus at its maximum level, whereas others showed little or no expression. The bimodal expression indicated that a gene marked by the insertion Ω4473 was differentially expressed in two cell populations—one at high level, the other at low. The “high” regulatory state of individual developing cells was maintained at least 3 h in the sorted cells (17). A second Tn5lac insertion, called Ω4414, was found at a different position within the same operon as Ω4473 (15, 18). The critical role of this operon in development was indicated by the fact that either insertion causes more than a 1,000-fold reduction in the number of viable spores. The operon identified by Ω4473 and Ω4414, which was named dev, is developmentally regulated, and its expression begins around the time of aggregation (18).

Because dev mutants are defective in sporulation, and because expression of the dev operon is either high or low in individual cells later in development, we have tested the possibility that dev expression is related to spore localization within the fruiting body. To find which cells express dev during development, we created a transcriptional fusion between the dev operon and the gfp gene, which encodes the green fluorescence protein. The observed spatial distribution of fluorescence of this fusion strain, indeed, shows localization to the fruiting body, and no expression in the peripheral rods. Examination of many developmentally regulated genes revealed a clear pattern: early genes were equally expressed in peripheral rods and fruiting bodies, whereas late genes were preferentially expressed in the fruiting body. Moreover, all of the differentially expressed genes depend on C-signaling.

Development in submerged culture for 5 days. Each assay was made three times. The mean values for three independent assays with the standard deviations in parentheses are shown.

Acknowledgments

Abbreviations