Signal transduction and regulation of developmental progression in Myxococcus xanthus

M. xanthus is a bacterium that exhibits a social (multicellular) life cycle. Under nutrient-limiting conditions, a complex developmental program is induced wherein cells first aggregate into mounds of approximately 100 000 cells and then, within these mounds, differentiate into spores forming fruiting bodies. These processes are tightly controlled by a series of temporally regulated extracellular and intracellular signals that must be coordinated and integrated to ensure proper fruiting body formation and sporulation efficiency.

We and others have identified a series of mutants (espA, espC, redCDEF, hpk30 and todK) that yield a developmental phenotype in which aggregation and sporulation are accelerated relative to wild type. These mutants also result in fruiting bodies that are smaller, more numerous, and less organized than wild type and in the appearance of spores outside of the fruiting bodies. We hypothesize that these mutants are disrupted for proteins that mediate orderly progression through the developmental program by acting as checkpoints to repress developmental progression until certain conditions are met. In the absence of these inhibitory mechanisms, aggregation and sporulation are premature and are not coupled resulting in disorganized fruiting bodies.

Interestingly, these mutants all map to histidine protein kinase genes that are members of the two-component signal transduction (TCS) family. TCS systems are a major mechanism for mediating responses in prokaryotes. In a paradigm system, a signal sensed by a histidine protein kinase induces autophosphorylation on a conserved histidine residue. The phosphoryl group is then transferred to a conserved aspartate residue in a response regulator protein which mediates a response: typically changes in gene transcription or enzyme activity. Most commonly, genes encoding histidine kinase and response regulator protein pairs are adjacent in the genome.

The genetic organization and domain architecture of the histidine kinases that were identified as being involved in regulation of developmental progression in M. xanthus in is unusual. For instance, espA, espC, and hpk30, and todK are encoded as orphan genes such that the cognate partner is not readily identified, while redCDEF is a complex system consisting of two histidine kinases and two response regulators. In addition, analysis of the protein sequence of the histidine kinase and response regulator proteins indicates there are unusual domain arrangements in these proteins, such as the presence of unusual sensing domains, or conversely, lack of identifiable sensing and output domains.

We have been working to understand at a molecular level the unusual signal transduction pathways in each of the systems described below, and to determine how these pathways are integrated to control timing of the developmental program.

EspAB/PktA5/PktB8

Previous research identified the espAB/pktA5/pktB8 locus that controls the timing of developmental progression. Mutants in espA, a hybrid histidine kinase homolog, cause cells to aggregate and sporulate earlier than wild type and result in fruiting bodies that are small, numerous, and disorganized. A combination of in vitro phosphorylation analyses and in vivo genetic analyses indicates that appropriate control of developmental progression requires that EspA autophosphorylates on a conserved histidine residue and then transfers the phosphoryl group to its associated receiver domain. It is unclear, however, what output protein(s) are activated by this signal flow.

To gain insight as to how EspA could control developmental progression, we have examined the expression patterns of a series of developmental markers in espA mutants compared to wild type. These analyses suggest that in wild type cells EspA represses protein accumulation of MrpC, a key transcriptional regulator which triggers a cascade of gene expression and signalling events necessary for induction of aggregation and sporulation. We are currently working to understand specifically how EspA effects MrpC protein accumulation.

Genetic epistasis analyses suggest that EspA may be induced to relieve repression of MrpC accumulation by a signalling module consisting of EspB, a putative oligopeptide transport protein, and PktA5 and PktB8, two Ser/Thr protein kinases.

Interestingly, EspA contains a forkhead associated (FHA) domain- a protein interaction module that binds to phospho-threonine containing proteins. We have demonstrated that the FHA domain appears to be necessary to receive signals from one or more of EspB, PktA5, and PktB8. EspA thus seems to receive and integrate signals from serine/threonine kinases, and a transport protein.

RedCDEF

Recently we characterized four additional unusual twocomponent signal transduction proteins (RedC, RedD, RedE and RedF) that also modulate developmental progression. We have demonstrated that redC-F are encoded together in a large operon containing at least seven genes. RedC encodes a membrane-bound histidine kinase homolog, RedD encodes a protein with dual receiver domains, RedE encodes a histidine kinase with no obvious sensing domain, while RedF encodes a single receiver domain protein.

Using a combination of in vitro phosphorylation analyses and in vivo genetic epistasis analyses we have determined that the Red TCS system represents a novel signal transduction mechanism described in Fig. 1. Our data suggests several unique features: 1) RedC-F function as a "four component2 signal transduction system, 2) RedD response regulator is necessary to induce RedC to act as its kinase (see Fig. 1 legend), 3) RedE histidine kinase does not appear to autophosphorylate but instead accepts a phosphoryl group from RedD, and 4) RedE acts solely as a phosphatase on RedF.

Our model predicts that, like the EspA signal transduction system, the Red signal transduction system appears to integrate multiple signals to control developmental progression. We are currently working to understand the nature of the signals that stimulate the system as well as the means by which RedF specifi cally affects the developmental program.

EspC, TodK, and Hpk30

Mutants in three additional orphan histidine protein kinase genes also yield similar premature developmental phenotypes when mutated: espC, hpk30, and todK. We are currently employing similar methods as those described above to determine how these proteins sense and respond to signals to control progression through the developmental program.

Control of developmental progression

We are also exploring how the signal transduction systems described above are coordinated to control progression through the developmental program. We have analyzed double mutants between each combination of kinase mutants for epistasis. Our results indicate that EspA and EspC may function together in the same pathway since the double mutant phenocopies each single mutant. However, TodK and RedCDEF function in separate pathways from each other and from EspA/EspC, since these combinations of double mutants display additively faster development. Taken together, these results suggest that there are at least three independent signalling pathways that control developmental progression. We have also determined that mutants lacking three or four of these kinases genes develop progressively additively faster such that spores can be produced as much as 36 hours earlier than wild type. Interestingly, there is a correlation between rate of development and loss of coordinated fruiting body formation; the fastest developing mutants form shallow layers of spores rather than well-rounded fruiting bodies. Our results suggest that coordinated social behaviour in M. xanthus is facilitated by these two-component signalling systems which appear to act in successive stages during the developmental program.

Spore Morphogenesis

We are also interested in the unique spore formation process in M. xanthus, one of the rare Gram negative spore-forming bacteria. Unlike spores produced from unequal cell division in Gram positive species, M. xanthus spores are produced by rounding up of the entire rod-shaped cell. Furthermore, it is not fully clear how environmental resistance is mediated, or how the spore coat is deposited outside of the outer membrane. To identify genes that play a role specifi cally in the sporulation process, we have performed microarray analysis on cells induced to form “quick spores” by addition of certain chemicals to vegetative cultures. In this alternative sporulation pathway, cells are rapidly and synchronisticly converted into resistant spores, bypassing the lengthy social behaviours involved in fi rst generating fruiting bodies.

By this approach, we identifi ed an operon of eight genes (termed K5) that, when deleted, rendered the cells unable to form resistant spores by either the starvation- or chemical-induced pathways. Interestingly, the delta-K5 mutant appears to have defects in rod/sphere shape transition: cells are unable to form spheres under the chemical induced sporulation pathway, instead forming ovoids. Furthermore, these ovoids eventually re-elongate forming transient spiral-like cells.

Bioinformatics analyses suggest these genes contain no domains of known function, but are predicted to form a complex in the cell envelope. We are currently investigating the hypothesis that, during sporulation and/or germination, these genes function to coordinate cell wall synthesis to cytoskeleton rearrangements.