Making waves: pattern formation by a cell-surface-associated signal

Starving Myxococcus xanthus cells organize into two strikingly different spatio-temporal patterns, either rippling or aggregation of cells into fruiting bodies. Formation of both patterns depends on a cell-surface-associated, non-diffusible signal, the C-signal. A key motility parameter modulated by the C-signal during pattern formation is the frequency at which cells reverse their gliding direction, with low and high levels of C-signalling causing an increase and a decrease in the reversal frequency, respectively. Recently, a simple yet elegant mathematical model was proposed to explain the mechanism underlying the non-linear dependence of the reversal frequency on C-signalling levels. The mathematical solution hinges on the introduction of a negative feedback loop into the biochemical circuit that regulates the reversal frequency. This system displays an oscillatory behaviour in which the oscillation frequency depends in a non-monotonic manner on the level of C-signalling. Thus, the biochemical oscillator recapitulates the effect of the C-signal on the reversal frequency. The challenge for biologists now is to test the mathematical model experimentally.     

Coupling of multicellular morphogenesis and cellular differentiation by an unusual hybrid histidine protein kinase in Myxococcus xanthus

We describe an unusual hybrid histidine protein kinase, which is important for spatially coupling cell aggregation and sporulation during fruiting body formation in Myxococcus xanthus. A rodK mutant makes abnormal fruiting bodies and spores develop outside the fruiting bodies. RodK is a soluble, cytoplasmic protein, which contains an N-terminal sensor domain, a histidine protein kinase domain and three receiver domains. In vitro phosphorylation assays showed that RodK possesses kinase activity. Kinase activity is essential for RodK function in vivo. RodK is present in vegetative cells and remains present until the late aggregation stage, after which the level decreases in a manner that depends on the intercellular A-signal. Genetic evidence suggests that RodK may regulate multiple temporally separated events during fruiting body formation including stimulation of early developmental gene expression, inhibition of A-signal production and inhibition of the intercellular C-signal transduction pathway. We speculate that RodK undergoes a change in activity during development, which is reflected in changes in phosphotransfer to the receiver domains.     

A sigma(54) activator protein necessary for spore differentiation within the fruiting body of Myxococcus xanthus

Insertion of an internal DNA fragment into the act1 gene, which encodes one of several sigma(54)-activator proteins in Myxococcus xanthus, produced a mutant defective in fruiting body development. While fruiting-body aggregation appears normal in the mutant, it fails to sporulate (<10(-6) the wild-type number of viable spores). The A and C intercellular signals, which are required for sporulation, are produced by the mutant. But, while it produces A-factor at levels as high as that of the wild type, the mutant produces much less C-signal than normal, as measured either by C-factor bioassay or by the total amount of C-factor protein detected with specific antibody. Expression of three C-factor-dependent reporters is altered in the mutant: the level of expression of Omega4414 is about 15% of normal, and Omega4459 and Omega4403 have alterations in their time course. Finally, the methylation of FrzCD protein is below normal in the mutant. It is proposed that Act1 protein responds to C-signal reception by increasing the expression of the csgA gene. This C-signal-dependent increase constitutes a positive feedback in the wild type. The act1 mutant, unable to raise the level of csgA expression, carries out only those developmental steps for which a low level of C-signaling is adequate.     

Cell behavior and cell-cell communication during fruiting body morphogenesis in Myxococcus xanthus

Formation of spatial patterns of cells from a mass of initially identical cells is a recurring theme in developmental biology. The dynamics that direct pattern formation in biological systems often involve morphogenetic cell movements. An example is fruiting body formation in the gliding bacterium Myxococcus xanthus in which an unstructured population of identical cells rearranges into an asymmetric, stable pattern of multicellular fruiting bodies in response to starvation. Fruiting body formation depends on changes in organized cell movements from swarming to aggregation. The aggregation process is induced and orchestrated by the cell-surface associated 17 kDa C-signal protein. C-signal transmission depends on direct contact between cells. Evidence suggests that C-signal transmission is geometrically constrained to cell ends and that productive C-signal transmission only occurs when cells engage in end-to-end contacts. Here, we review recent progress in the understanding of the pattern formation process that leads to fruiting body formation. Gliding motility in M. xanthus involves two polarly localized gliding machines, the S-machine depends on type IV pili and the A-machine seems to involve a slime extrusion mechanism. Using time-lapse video microscopy the gliding motility parameters controlled by the C-signal have been identified. The C-signal induces cells to move with increased gliding speeds, in longer gliding intervals and with decreased stop and reversal frequencies. The combined effect of the C-signal dependent changes in gliding motility behaviour is an increase in the net-distance travelled by a cell per minute. The identification of the motility parameters controlled by the C-signal in combination with the contact-dependent C-signal transmission mechanism have allowed the generation of a qualitative model for C-signal induced aggregation. In this model, the directive properties of the C-signal are a direct consequence of the contact-dependent signal-transmission mechanism, which is a local event involving direct contact between cells that results in a global organization of cells. This pattern formation process does not depend on a diffusible substance. Rather it depends on a cell-surface associated signal to direct the cells appropriately.     

Cell polarity, intercellular signalling and morphogenetic cell movements in Myxococcus xanthus

In Myxococcus xanthus morphogenetic cell movements constitute the basis for the formation of spreading vegetative colonies and fruiting bodies in starving cells. M. xanthus cells move by gliding and gliding motility depends on two polarly localized engines, type IV pili pull cells forward, and slime extruding nozzle-like structures appear to push cells forward. The motility behaviour of cells provides evidence that the two engines are localized to opposite poles and that they undergo polarity switching. Several proteins involved in regulating polarity switching have been identified. The cell surface-associated C-signal induces the directed movement of cells into nascent fruiting bodies. Recently, the molecular nature of the C-signal molecule was elucidated and the motility parameters regulated by the C-signal were identified. From the effect of the C-signal on cell behaviour it appears that the C-signal inhibits polarity switching of the two motility engines. This establishes a connection between cell polarity, signalling by an intercellular signal and morphogenetic cell movements during fruiting body formation.     

The act operon controls the level and time of C-signal production for Myxococcus xanthus development

The C-signal is a morphogen that controls the assembly of fruiting bodies and the differentiation of myxospores. Production of this signal, which is encoded by the csgA gene, is regulated by the act operon of four genes that are co-transcribed from the same start site. The act A and act B genes regulate the maximum level of the C-signal, which never rises above one-quarter of the maximum wild-type level of CsgA protein in null mutants of either gene. The act A and act B mutants have the same developmental phenotype: both aggregate, neither sporulates, both prolong rippling. By sequence homology, act A encodes a response regulator, and act B encodes a sigma-54 activator protein of the NTRC class. The similar phenotypes of act A and act B deletion mutants suggest that the two gene products are part of the same signal transduction pathway. That pathway responds to C-signal and also regulates the production of CsgA protein, thus creating a positive feedback loop. The act C and act D genes regulate the time pattern of CsgA production, while achieving the same maximum level. An act C null mutant raises CsgA production 15 h earlier than the wild type, whereas an act D null mutant does so 6 h later than wild type. The loop explains how the C-signal rises continuously from early development to a peak at the time of sporulation, and the act genes govern the time course of that rise.     

Regulated secretion of a protease activates intercellular signaling during fruiting body formation in M. xanthus

In response to starvation Myxococcus xanthus initiates a developmental program that culminates in fruiting body formation. There are two morphogenetic events in this program, aggregation and sporulation, which are temporally and spatially coordinated by the contact-dependent intercellular C-signal protein (p17). p17 is generated by proteolytic cleavage of the p25 precursor protein, which accumulates in the outer membrane of vegetative and starving cells. However, p17 generation is restricted to starving cells. Here we identify the subtilisin-like protease PopC that is directly responsible for cleavage of p25. PopC accumulates in the cytoplasm of vegetative cells but is selectively secreted during starvation coinciding with the generation of p17. Consequently, p25 and PopC only encounter each other in starving cells. Thus, restriction of p25 cleavage to starving cells occurs by a compartmentalization mechanism that depends on selective secretion of PopC during starvation. Our results provide evidence for regulated proteolysis via regulated secretion.     

Pattern formation: fruiting body morphogenesis in Myxococcus xanthus

When Myxococcus xanthus cells are exposed to starvation, they respond with dramatic behavioral changes. The expansive swarming behavior stops and the cells begin to aggregate into multicellular fruiting bodies. The cell-surface-associated C-signal has been identified as the signal that induces aggregation. Recently, several of the components in the C-signal transduction pathway have been identified and behavioral analyses are beginning to reveal how the C-signal modulates cell behavior. Together, these findings provide a framework for understanding how a cell-surface-associated morphogen induces pattern formation.     

Pattern formation: fruiting body morphogenesis in Myxococcus xanthus

When Myxococcus xanthus cells are exposed to starvation, they respond with dramatic behavioral changes. The expansive swarming behavior stops and the cells begin to aggregate into multicellular fruiting bodies. The cell-surface-associated C-signal has been identified as the signal that induces aggregation. Recently, several of the components in the C-signal transduction pathway have been identified and behavioral analyses are beginning to reveal how the C-signal modulates cell behavior. Together, these findings provide a framework for understanding how a cell-surface-associated morphogen induces pattern formation.     

Developmental bypass suppression of Myxococcus xanthus csgA mutations.

The csgA mutations of Myxococcus xanthus (formerly known as spoC) inhibit sporulation as well as rippling, which involves ridges of cells moving in waves. Sporulating revertants of CsgA cells were isolated by direct selection, since spores are much more resistant to heat and ultrasonic treatment than are vegetative cells. The revertants fell into seven groups on the basis of phenotype and the chromosomal location of the suppressor alleles. Group 1 contained one allele that was a back mutation of the original csgA mutation. Group 2 contained two linked alleles that were unlinked to the csgA locus and restored fruiting-body formation, sporulation, and rippling. Group 3 revertants regained the ability to sporulate in fruiting bodies but not the ability to ripple. Revertants in groups 4 to 7 were able to sporulate but unable to form fruiting bodies or ripples. The suppressors were all found to be bypass suppressors even though they were not selected as such in most cases. The csgA mutation prevented expression of several developmentally regulated promoters, each fused to a lacZ reporter gene and assayed by beta-galactosidase production. In four of five suppressor groups (groups 4 to 7), expression of each of these csgA-dependent fusions was restored, which suggests that bypass suppression restores developmental gene expression near the point at which expression is disrupted in CsgA mutants. Bypass suppression did not restore production of C factor, and morphological manifestations of development such as rippling and fruiting-body formation were usually abnormal. One interpretation of these results is that C factor has multiple functions and few suppressors can compensate for all of them.     

C-factor: a cell-cell signaling protein required for fruiting body morphogenesis of M. xanthus

During fruiting body development, the product of the csgA gene is necessary for cellular aggregation, for spore differentiation, and for gene expression that is initiated after 6 hr of starvation. From nascent wild-type fruiting bodies we have purified a polypeptide of 17 kd called C-factor, which, at approximately 1 to 2 nM, restores normal development to csgA mutant cells. C-factor activity is not recovered from extracts of unstarved, growing cells or csgA mutant cells. The amino acid sequence from purified C-factor demonstrates that it is the product of the csgA gene. C-factor is active over a narrow range of concentration and has properties of a morphogenetic paracrine signal.     

Pleurotus and Agrocybe hemolysins,new proteins hypothetically involved in fungal fruiting

Novel hemolytic proteins, ostreolysin and aegerolysin, were purified from the fruiting bodies of the edible mushrooms Pleurotus ostreatus and Agrocybe aegerita. Both ostreolysin and aegerolysin have a molecular weight of about 16 kDa, have low isoelectric points of 5.0 and 4.85, are thermolabile, and hemolytic to bovine erythrocytes at nanomolar concentrations. Their activity is impaired by micromolar Hg(2+) but not by membrane lipids and serum low-density lipoproteins (LDL). The sequence of respectively 50 and 10 N-terminal amino acid residues of ostreolysin and aegerolysin has been determined and found to be highly identical with a cDNA-derived amino acid sequence of putative Aa-Pri1 protein from the mushroom A. aegerita, Asp-hemolysin from Aspergillus fumigatus, and two bacterial hemolysin-like proteins expressed during sporulation. We found that ostreolysin is expressed during formation of primordia and fruiting bodies, which is in accord with previous finding that the Aa-Pri1 gene is specifically expressed during fruiting initiation. It is suggestive that the isolated hemolysins play an important role in initial phase of fungal fruiting.     

EspC is involved in controlling the timing of development in Myxococcus xanthus

The espC null mutation caused accelerated aggregation and formation of tiny fruiting bodies surrounded by spores, which were also observed in the espA mutant and in CsgA-overproducing cells in Myxococcus xanthus. In addition, the espC mutant appeared to produce larger amounts of the complementary C-signal than the wild-type strain. These findings suggest that EspC is involved in controlling the timing of fruiting body development in M. xanthus.