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Ultrasensitive Response of Developing Myxococcus Xanthus to the Addition of Nutrient Medium Correlates With the Level of MrpC

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Ultrasensitive Response of Developing Myxococcus Xanthus to the Addition of Nutrient Medium Correlates With the Level of MrpC

Y Hoang et al. J Bacteriol.

Abstract

Upon depletion of nutrients, Myxococcus xanthus forms mounds on a solid surface. The differentiation of rod-shaped cells into stress-resistant spores within mounds creates mature fruiting bodies. The developmental process can be perturbed by the addition of nutrient medium before the critical period of commitment to spore formation. The response was investigated by adding a 2-fold dilution series of nutrient medium to starving cells. An ultrasensitive response was observed, as indicated by a steep increase in the spore number after the addition of 12.5% versus 25% nutrient medium. The level of MrpC, which is a key transcription factor in the gene regulatory network, correlated with the spore number after nutrient medium addition. The MrpC level decreased markedly by 3 h after adding nutrient medium but recovered more after the addition of 12.5% than after 25% nutrient medium addition. The difference in MrpC levels was greatest midway during the period of commitment to sporulation, and mound formation was restored after 12.5% nutrient medium addition but not after adding 25% nutrient medium. Although the number of spores formed after 12.5% nutrient medium addition was almost normal, the transcript levels of "late" genes in the regulatory network failed to rise normally during the commitment period. However, at later times, expression from a reporter gene fused to a late promoter was higher after adding 12.5% than after adding 25% nutrient medium, consistent with the spore numbers. The results suggest that a threshold level of MrpC must be achieved in order for mounds to persist and for cells within to differentiate into spores.IMPORTANCE Many signaling and gene regulatory networks convert graded stimuli into all-or-none switch-like responses. Such ultrasensitivity can produce bistability in cell populations, leading to different cell fates and enhancing survival. We discovered an ultrasensitive response of M. xanthus to nutrient medium addition during development. A small change in nutrient medium concentration caused a profound change in the developmental process. The level of the transcription factor MrpC correlated with multicellular mound formation and differentiation into spores. A threshold level of MrpC is proposed to be necessary to initiate mound formation and create a positive feedback loop that may explain the ultrasensitive response. Understanding how this biological switch operates will provide a paradigm for the broadly important topic of cellular behavior in microbial communities.

Keywords: FruA; MrpC; Myxococcus xanthus; bacterial development; gene regulatory network; sporulation; ultrasensitive response.

Figures

FIG 1
FIG 1
Simplified model of the gene regulatory network governing M. xanthus development. Starvation increases the level of phosphorylated MrpB (MrpB-P), which activates mrpC transcription. MrpC negatively autoregulates by competing with MrpB-P for binding to the mrpC promoter region. The addition of nutrient medium can halt development by lowering the mrpC transcript level (as described in this study but not depicted in the model) and by inducing proteolysis of MrpC, as depicted here. MrpC activates transcription of the gene for FruA and causes an increase in C-signaling, which posttranslationally activates FruA to FruA*. FruA* promotes mound formation, which enhances short-range C-signaling by bringing cells into proximity, creating a positive feedback loop. FruA* and MrpC bind cooperatively to the promoter region of the dev operon and activate transcription. The resulting DevTRS proteins negatively autoregulate. DevI normally delays cellular shape change leading to spore maturation, but if overproduced, DevI blocks sporulation. A rising level of FruA* in cells within mounds, together with MrpC, activates the transcription of genes involved in commitment to cellular shape change and spore maturation, resulting in spore-filled fruiting bodies. See the text for details and references.
FIG 2
FIG 2
The ultrasensitive response of developing M. xanthus to nutrient medium addition. Wild-type strain DK1622 was subjected to starvation under submerged culture conditions. The culture supernatant was replaced with the indicated percentage of CTTYE nutrient medium at 18 h poststarvation (PS). (A) Effect of CTTYE addition on fruiting body formation. Images were obtained at 18 h PS and at the indicated times of total incubation. Mounds formed by 18 h PS (an arrow points to one) and darkened by 27 h of total incubation when the culture supernatant was replaced with fresh starvation buffer (0% CTTYE). Cells dispersed from mounds when the culture supernatant was replaced with CTTYE, but mounds reformed by 27 h of total incubation with 12.5% CTTYE, whereas cells remained dispersed with 25% or 100% CTTYE. Bar = 100 μm. Similar results were observed in three biological replicates. (B) Effect of CTTYE addition on sonication-resistant spore formation. Cultures were harvested at the indicated times of total incubation for the measurement of sonication-resistant spores. Log-log plot shows the average of three biological replicates (y axis; error bars show one standard deviation) at each nutrient concentration (x axis).
FIG 3
FIG 3
Effect of nutrient medium addition on formation of sonication-resistant spores and mature spores. Wild-type M. xanthus strain DK1622 was subjected to starvation under submerged culture conditions. The culture supernatant was replaced with fresh starvation buffer (0% CTTYE) or the indicated percentage of CTTYE nutrient medium at 18 h poststarvation, and cultures were harvested at the indicated times of total incubation for the measurement of sonication-resistant spores and mature spores. Values are the average of three biological replicates, and error bars show one standard deviation.
FIG 4
FIG 4
Effect of nutrient medium addition on the mrpC transcript and MrpC protein levels. Wild-type M. xanthus strain DK1622 was subjected to starvation under submerged culture conditions. At 18 h poststarvation, culture supernatants were replaced with fresh starvation buffer (0%) or the percentage of CTTYE nutrient medium indicated in the key. Cultures were harvested at the indicated times of total incubation. (A) mrpC transcript levels. RNA was isolated from the cultures and subjected to quantitative reverse transcription-PCR (RT-qPCR) analysis. (B) MrpC protein levels. Protein samples from the cultures were analyzed by immunoblotting using anti-MrpC antibodies. In both panels, values are the average of three biological replicates, relative to the sample at 18 h, and error bars show one standard deviation.
FIG 5
FIG 5
Effect of nutrient medium addition on the mrpB transcript level. Wild-type M. xanthus strain DK1622 was subjected to starvation under submerged culture conditions. At 18 h poststarvation, culture supernatants were replaced with fresh starvation buffer (0%) or the percentage of CTTYE nutrient medium indicated in the key. Cultures were harvested at the indicated times of total incubation. RNA was isolated from the cultures and subjected to RT-qPCR analysis. Values are the average of three biological replicates, relative to the sample at 18 h, and error bars show one standard deviation.
FIG 6
FIG 6
MrpC protein stability after nutrient medium addition. Wild-type M. xanthus strain DK1622 was subjected to starvation under submerged culture conditions in 6-well plates. At 18 h poststarvation, culture supernatants were replaced with fresh starvation buffer (0%) or the percentage of CTTYE nutrient medium indicated in the key. At 27 h of total incubation, culture supernatants were supplemented with 200 μg/ml chloramphenicol, and a culture was harvested immediately (t0) and at each indicated time (tx) after chloramphenicol addition, for measurement of the MrpC level by immunoblot using anti-MrpC antibodies. MrpC levels at tx were normalized to that at t0 for each of three biological replicates and used to determine the MrpC half-life for each replicate. The average half-life (average t1/2) and one standard deviation are shown in the legend. The graph shows the average ln(tx/t0) and one standard deviation for the three biological replicates.
FIG 7
FIG 7
Effect of nutrient medium addition on the fruA transcript and FruA protein levels. Wild-type M. xanthus strain DK1622 was subjected to starvation under submerged culture conditions. At 18 h poststarvation, culture supernatants were replaced with fresh starvation buffer (0%) or the percentage of CTTYE nutrient medium indicated in the key. Cultures were harvested at the indicated times of total incubation. (A) fruA transcript levels. RNA was isolated from the cultures and subjected to RT-qPCR analysis. (B) FruA protein levels. Protein samples from the cultures were analyzed by immunoblotting using anti-FruA antibodies. In both panels, values are the average of three biological replicates, relative to the sample at 18 h, and error bars show one standard deviation.
FIG 8
FIG 8
Effect of nutrient medium addition on late gene transcript levels. Wild-type M. xanthus strain DK1622 was subjected to starvation under submerged culture conditions. At 18 h poststarvation, culture supernatants were replaced with fresh starvation buffer (0%) or the percentage of CTTYE nutrient medium indicated in the key. Cultures were harvested at the indicated times of total incubation. RNA was isolated from the cultures and subjected to RT-qPCR analysis. Values are the average of three biological replicates, relative to the sample at 18 h, and error bars show one standard deviation. (A and B) dev (A) and exo (B) transcript levels.
FIG 9
FIG 9
Effect of nutrient medium addition on exo-lacZ expression. M. xanthus strain DK10524 was subjected to starvation under submerged culture conditions in 6-well plates. At 18 h poststarvation, culture supernatants were replaced with fresh starvation buffer (0%) or the percentage of CTTYE nutrient medium indicated in the key. Cultures were harvested at the indicated times of total incubation for the measurement of β-galactosidase activity. Values are the average of three biological replicates, and error bars show one standard deviation.

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