The Prokaryotic Development and Differentiation Group

Research Program

Signal transduction pathways in prokaryotic development and differentiation

Current research interests

  • cellular differentiation in prokaryotes
  • multicellular development in prokaryotes
  • intercellular signaling
  • signal transduction mechanisms and - pathways
  • protein-protein interactons in signal transduction pathways
  • developmentally regulated gene expression (e.g. gene regulatory switches that are regulated by morphological checkpoints)
  • pattern formation
  • cellular organization

Current reseach activities

Signal transduction in multicellular devlopment and cellular differention: the C-factor signal transduction pathway in M.xanthus.

Multicellular development in M.xanthus: fruiting body morphogenesis

The grand theme in our research is "intercellular communication by signal molecules in the induction and coordination of multicellular development and cellular differentiation". We have focused on understanding how a particular intercellular signal molecule in the Gram negative bacterium Myxococcus xanthus induces a set of different responses during starvation induced development, including changed motility patterns, sporulation and altered gene expression. M. xanthus undergoes one of the most astonishing prokaryotic developmental programs upon starvation. Within 4 to 8 hrs after initiation of starvation, the cells begin to aggregate by gliding to foci where 105 cells build a fruiting body. Inside a fruiting body, the rod-shaped, motile cells differentiate into spherical, non-motile spores by approximately 24 hrs. Aggregation and sporulation are temporally separated, and sporulation does not occur until cell migration have led to the assembly of a fruiting body. So, initiation of sporulation represents a developmental checkpoint at which cellular differentiation is coupled to the morphogenesis of a multicellular structure.

C-factor signaling

Fruiting body morphogenesis in M. xanthus is controlled by at least five intercellular signals. Our research focus on understanding signaling by C-factor. C-factor is a 17 kD cell-surface associated protein and encoded by the csgA gene. C-factor signaling begins after about 6 hrs of starvation and induces aggregation, sporulation, csgA expression and developmentally regulated gene expression. Thus, C-factor is a key signaling molecule that directly induces the two major morphological events during fruiting body morphogenesis, aggregation and sporulation. In the presence of trace amounts of nutrients, C-factor is required for a different type of organized cell movements called ripples. In this case, the cells rather than aggregating to form fruiting bodies, form ridges of cells that move like travelling waves on a surface. So, there are at least four C-factor dependent responses

  • A change in motility pattern: from swarming to aggregation or rippling;
  • Cellular differentiation: sporulation;
  • Developmentally regulated gene expression including induction of csgA expression.

A model for the C-factor signal transduction pathway

Our research is aimed at understanding how C-factor is recognized and how the C-factor signaling pathway is structured to provide qualitatively different and temporally separated responses. Using a genetic approach we have isolated mutants deficient in some or all of the C-factor responses. Genetic analysis of these mutants allowed the construction of a model for this pathway:

The activating signaling event in the pathway is the interaction between C-factor on one cell-end with a C-factor receptor on a second cell-end. The class II component is the earliest acting known component in the pathway and is required for all C-factor dependent responses. The C-factor signal is transduced from the product of the class II gene to the Frz proteins in a branch that leads to the motility response to C-factor; a second branch from class II leads to the sporulation, csgA expression and developmental gene expression response. The devRS operon is a component in this pathway.

The motility branch in the C-factor signal transduction pathway: the C-factor-classII-Frz connection

The Frz proteins constitute a cytoplasmic signal transduction system that controls the frequency of gliding reversals in response to chemical stimuli. It consists of at least six proteins, five of which share homology with chemotaxis two-component regulatory systems in enteric bacteria. We have shown that C-factor stimulate the Frz system in a class II dependent fashion as monitored by an increased level of methylation of FrzCD protein in response to exogenous C-factor. These results firmly establish a connection between the cell-surface C-factor signal transmission and the cytoplasmic Frz system and they suggest that the class II component is the mediator between the two compartments.

A model for C-factor induced aggregation:

On the basis of these results, we have proposed a model for how a cell-bound intercellular signal like C-factor can promote aggregation. The fundamental cell-cell interaction in this model is an end-to-end contact with C-factor signaling between two cells. The signaling event stimulates the Frz system and confers the capacity to move with a low reversal frequency onto the signaling cells. Since the ability to move with the low reversal frequency depends on continuous C-factor signaling, only cells that maintain the end-to-end contact maintain the capacity to move with the low reversal frequency. Applied to a field of starving cells, this model provides a logic to explain aggregation. The model predicts that cells are recruited sequentially to the ends of chains of cells that are moving towards aggregation centers. The C-factor signal is relayed from the aggregation center to the end of the chain by end-to-end contacts. This model which we call "chemotaxis towards a cell-bound attractant" represent a novel mechanism for how organized cell migration can be obtained and may prove to be of fundamental importance in understanding organized cell migration and pattern formation in other systems.

The sporulation branch in the C-factor signal transduction pathway: the C-factor-classII-devRS connection

devRS mutants are deficient in sporulation, however, they aggregate normally. In a csgA mutant, expression of the devRS locus is reduced as measured by using Tn5 lac inserted in devS. Using the same experimental setup, we have shown that in a class II mutant, expression of a devS::Tn5 lac fusion is reduced. So, C-factor signaling results in a class II dependent increase in devRS expression. In addition, C-factor production is reduced in a devS mutant. On the basis of these results, a simple model for the interaction between C-factor and devRS would be that C-factor signaling activates the class II component. Subsequently, this activation results in increased expression of devRS.

Experimental plans

Our models for the C-factor signal transduction pathway and for C-factor signaling induced aggregation in combination with the identification of several of the components in this pathway have opened the road to a comprehensive and systematic identification of additional components in the pathway and to analyses of how these components interact. Using a combination of genetic, molecular biology, biochemical, mass spectrometry and cell imaging approaches, we are focusing on the following project

  • Identification of the C-factor receptor;
  • Cloning and characterization of class II component(s);
  • Characterization of how the class II component and the Frz proteins interact;
  • Identification of proteins that act downstream of the class II component in the sporulation/gene expression branch;
  • Identification of cis-acting sequences, and trans-acting factors involved in devRS expression;
  • Determination of the cellular and subcellular localization of C-factor and its receptor;
  • Determination of the behavioural response of cells to C-factor signaling;
  • Characterization of the active form of C-factor.

   

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