Autocide AMI rescues development in dsg mutants of Myxococcus xanthus

Low concentrations of autocide AMI rescued aggregation and sporulation in the dsg mutant class of Myxococcus xanthus but were incapable of rescuing asg, bsg, or csg mutants. AMI-induced spores of dsg mutants were resistant to heat and sonication and germinated when plated on nutrient-rich agar. AMI accelerated aggregation and sporulation and increased the final spore number in submerged cultures of a wild-type strain of M. xanthus. Development of M. xanthus was accompanied by release of a fluorescent material (emission maximum, 438 nm) into the supernatant fluid. The release of this material began early and continued throughout development. All Spo- mutant strains tested released significantly reduced levels of this material. These levels were increased in the presence of AMI in all Spo- mutant classes, most dramatically in the dsg mutants.     

dsg, a gene required for cell-cell interaction early in Myxococcus development.

dsg mutants of Myxococcus xanthus are conditionally defective in fruiting body development, including sporulation. Unable to develop on their own, these mutants can assemble fruiting bodies with spores if they are mixed with wild-type cells. To elucidate the developmental defect in dsg mutants by close comparison with wild type, such mutants have been backcrossed by transduction, using a closely linked insertion of transposon Tn5 for selection. Backcrossed dsg mutants form aggregates that are larger, less compact, and less symmetrical than dsg+ fruiting bodies. Also, the starvation-induced sporulation in dsg aggregates is delayed and reduced. However, dsg mutants can be induced by glycerol or dimethyl sulfoxide to sporulate at levels approaching those of wild type. dsg mutants may thus have a primary defect early in development which diminishes their capacity to aggregate and which indirectly decreases the number of fruiting body spores. The linked insertion of Tn5 also facilitated cloning the dsg gene. The cloned dsg+ allele was shown to be dominant to both the dsg-429 and dsg-439 alleles, and both mutant alleles were shown to belong to the same genetic complementation group. Subcloning of restriction fragments, deletions, and insertions of transposon Tn5 agree in locating the dsg gene to an 850-base-pair segment of the cloned region.     

Myxococcus xanthus autocide AMI.

Autocide AMI of Myxococcus xanthus was purified and shown to be a mixture of fatty acids: 46.4% saturated, 49.3% monounsaturated, and 4.3% diunsaturated. The specific autocidal activities (units per milligram) were as follows: purified AMI, 1,000; saturated fraction, 100; monounsaturated fraction, 800; diunsaturated fraction, 2,200. Model fatty acids mimicked to some extent the activity of AMI, although none of the fatty acids tested were as active as purified AMI. Spontaneous and induced mutants of M. xanthus were selected for resistance to AMI and to fatty acids. The AMI-resistant mutants were also resistant to the model fatty acids, whereas resistance to fatty acids was specific to the compound used for mutant selection. All AMI- and fatty acid-resistant mutants examined were found to be blocked in fruiting body formation. Some of these mutants were able to form normal fruiting bodies when mixed with the extracellular fluid of the parental strain. The data suggest that AMI plays a role in developmental lysis of M. xanthus.     

An endo-N-acetyl-beta-D-glucosaminidase, acting on the di-N-acetylchitobiosyl part of N-linked glycans, is secreted during sporulation of Myxococcus xanthus.

After the demonstration that Stigmatella aurantiaca DW4 secretes an endo-N-acetyl-beta-D-glucosaminidase (ENGase), acting on the di-N-acetylchitobiosyl part of N-linked glycans (S. Bourgerie, Y. Karamanos, T. Grard, and R. Julien, J. Bacteriol. 176:6170-6174, 1994), an ENGase activity having the same substrate specificity was also found to be secreted during vegetative growth of Myxococcus xanthus DK1622. The activity decreased in mutants known to secrete less protein than the wild type (Exc +/-). During submerged development, the activity was produced in two steps: the first increase occurred during the aggregation phase, and the second one occurred much later, during spore formation. This production was lower in developmental mutants impairing cell-cell signaling, the late mutants (csg and dsg) being the most deficient. Finally, when sporulation was obtained either by starvation in liquid shake flask culture or by glycerol induction, the activity was produced exclusively by the wild-type cells during the maturation of the coat.     

A development-specific protein in Myxococcus xanthus is associated with the extracellular fibrils.

We have been using monoclonal antibodies (MAbs) as probes to study developmentally relevant cell surface antigens (CSA) that may be required for cellular interactions in Myxococcus xanthus. Three independently isolated MAbs, G69, G357, and G645, isolated by Gill and Dworkin recognize a CSA detectable only on developing cells (J. S. Gill and M. Dworkin, J. Bacteriol. 168:505-511, 1986). The CSA is made within the first 30 min of submerged development and increases until myxosporulation. The CSA is also produced at low levels after 24 h in shaken-starved cultures and during glycerol sporulation. No antigen can be detected in lysed, vegetative cells, and expression of the antigen is blocked in the presence of rifampin or chloramphenicol. The antigen is expressed in submerged, developmental cultures of asg, bsg, csg, dsg, and mgl mutants and is not expressed in a dsp mutant. All of the three MAbs immunoprecipitate the same protein of approximately 97,000 Da from lysed developmental cells. Competitive immunoprecipitations suggest that they recognize at least two different epitopes on the CSA. The epitopes recognized by MAbs G69, G357, and G645 are sensitive to protease digestion, whereas the epitopes recognized by MAbs G357 and G645 are resistant to periodate oxidation. The epitope recognized by MAb G69 is sensitive to periodate oxidation. Fractionation of lysed developing cells shows that most of the antigen is localized in the pellet after centrifugation at 100,000 x g. To determine whether the antigen is expressed on the cell surface, we labeled developing whole cells with either MAb G69, G357, or G645 and gold-labeled anti-mouse immunoglobulin G. Low-voltage scanning electron microscopy of labeled cells shows that the antigen is associated with the fibrillar matrix that surrounds the cells and that the antigen is retained on isolated, developmental fibrils from M. xanthus. The CSA has been designated dFA-1, for developmental fibrillar antigen 1.     

Production of an extracellular milk-clotting activity during development in Myxococcus xanthus.

We describe here an extracellular proteolytic activity secreted during both growth and submerged development by Myxococcus xanthus DK1622. This activity yields the clotting of kappa-casein at pH 6 and is inhibited by specific inhibitors of aspartic proteases. Secretion of this milk-clotting proteolytic activity (of Mcp) is time regulated during the developmental cycle, with a large increase near 9 h poststarvation, but its production does not require cell-cell contact. The lack of secretion of this activity by several developmental mutants in submerged development conditions shows that Mcp production is developmentally regulated.     

Effects of glucosamine on lysis, glycerol formation, and sporulation in Myxococcus xanthus.

Glucosamine (GlcN), which has previously been shown to rescue fruiting body formation, lysis, and sporulation in a developmental mutant (G. Janssen and M. Dworkin, Dev. Biol. 112:194-202, 1985), induced lysis in vegetative and developing wild-type cells and inhibited fruiting body formation. It also resulted in a transient, intracellular increase in the concentration of glycerol, a known sporulation inducer, and sporulation of the surviving cells. Phospholipase activity, which was shown to be normally developmentally regulated, increased 7.6-fold after treatment of vegetative cells with 50 mM GlcN. Likewise, autocidal activity, which normally increased 18 to 24 h after the initiation of development, increased 20% when vegetative or developing cells were exposed to GlcN. Two mutants resistant to GlcN-induced lysis (MD1021 and MD1022) were isolated and showed neither an increase in autocide production nor an increase in phospholipase activity in response to added GlcN. MD1021 was developmentally deficient, and GlcN rescued fruiting body formation as well as phospholipase activity and autocide production. We propose that GlcN exerts its lytic effect by regulating the activity of phospholipase enzymes that release autocides, compounds that are believed to be responsible for developmental autolysis. GlcN-induced sporulation was found to depend on several factors: the initial cell density, the amount of lysis induced by GlcN, and the presence of tan-phase variants. An initial cell density of greater than 2 x 10(5) cells per ml was required to support GlcN-induced sporulation, and sporulation did not occur unless 50 to 75% of these cells had lysed. Mutants that were resistant to GlcN-induced lysis also did not sporulate in the presence of GlcN. The effects of GlcN on developing cells depended on the concentration of GlcN added; the addition of low concentrations of GlcN resulted in enhancement of sporulation, while higher concentrations resulted in the inhibition of sporulation. The ultrastructure of GlcN-induced spores resembled that of spores induced by the exogenous addition of glycerol, in contrast to spores isolated from mature fruiting bodies. A model by which GlcN may regulate both lysis and sporulation is presented.     

Cell-cell interactions in developmental lysis of Myxococcus xanthus

The developmental events of sporulation and fruiting body formation in the prokaryote Myxococcus xanthus are preceded by a stage of massive cell death. Two phenotypically complementable strains of M. xanthus defective in developmental lysis were identified from a group of conditional sporulation mutants. Mixture of the two lysis groups resulted in full complementation of lysis, sporulation, and fruiting body formation; efficient sporulation was observed only in strain mixtures where lysis was complemented. We have identified a cell-free extract from developing cells that phenotypically complemented lysis, sporulation, and fruiting body formation in one group of mutants; the active component of this extract appeared to be tightly cell associated. The effect of the cell-free extract could be replaced by exogenously supplied glucosamine or mannosamine.     

Branched-chain fatty acids: the case for a novel form of cell-cell signalling during Myxococcus xanthus development

The esg locus is required for the formation of muiti-cellular fruiting bodies and spores by the developmental bacterium Myxococcus xanthus Studies have suggested that esg mutants are defective in the production of an essential signal (E-signal) used in cell-cell communication and that E-signalling is required for the expression of many developmental genes. Recently we have determined that the esg locus encodes components of a branched-chain keto acid dehydrogenase. a multienzyme complex involved in branched-chain amino acid metabolism in many bacteria and higher organisms. During vegetative growth in M. xanthus. this enzyme complex appears to participate in the production of the branched-chain fatty acids found in this organism. M. xanthus fatty acids (including the branched-chain fatty acids) have been observed to have a variety of effects on developing cells. These effects include; (i) the lysis of M. xanthus cells (autocide activity), (ii) acceleration of the rate of sporulation and (iii) rescue of sporulation by certain development-defective mutants. These and other results suggest a model in which the branched-chain fatty acids. Synthesized during growth, are released from cellular phospholipid by a developmentally regulated phospholipase during fruiting-body formation. This model proposes that one or more of the branched-chain fatty acids that are released constitutes the E-signal which must be transmitted between cells to complete M. xanthus development.     

The Myxococcus xanthus dsg gene product performs functions of translation initiation factor IF3 in vivo.

The amino acid sequence of the Dsg protein is 50% identical to that of translation initiation factor IF3 of Escherichia coli, the product of its infC gene. Anti-E. coli IF3 antibodies cross-react with the Dsg protein. Tn5 insertion mutations in dsg are lethal. When ample nutrients are available, however, certain dsg point mutant strains grow at the same rate as wild-type cells. Under the starvation conditions that induce fruiting body development, these dsg mutants begin to aggregate but fail to develop further. The level of Dsg antigen, as a fraction of total cell protein, does not change detectably during growth and development, as expected for a factor essential for protein synthesis. The amount of IF3 protein in E. coli is known to be autoregulated at the translational level. This autoregulation is lost in an E. coli infC362 missense mutant. The dsg+ gene from Myxococcus xanthus restores normal autoregulation to the infC362 mutant strain. Dsg is distinguished from IF3 of E. coli, other enteric bacteria, and Bacillus stearothermophilus by having a C-terminal tail of 66 amino acids. Partial and complete deletion of this tail showed that it is needed for certain vegetative and developmental functions but not for viability.     

Lipophilic regulator of a developmental switch in Caenorhabditis elegans

Abstract In Caenorhabditis elegans, the decision to develop into a reproductive adult or arrest as a dauer larva is influenced by multiple pathways including insulin-like and transforming growth factor beta (TGFbeta)-like signalling pathways. It has been proposed that lipophilic hormones act downstream of these pathways to regulate dauer formation. One likely target for such a hormone is DAF-12, an orphan nuclear hormone receptor that mediates these developmental decisions and also influences adult lifespan. In order to find lipophilic hormones we have generated lipophilic extracts from mass cultures of C. elegans and shown that they rescue the dauer constitutive phenotype of class 1 daf-2 insulin signalling mutants and the TGFbeta signalling mutant daf-7. These extracts are also able to rescue the lethal dauer phenotype of daf-9 mutants, which lack a P450 steroid hydroxylase thought to be involved in the synthesis of the DAF-12 ligand; extracts, however, have no effect on a DAF-12 ligand binding domain mutant that is predicted to be ligand insensitive. The production of this hormone appears to be DAF-9 dependent as extracts from a daf-9;daf-12 double mutant do not exhibit this activity. Preliminary fractionation of the lipophilic extracts shows that the activity is hydrophobic with some polar properties, consistent with a small lipophilic hormone. We propose that the dauer rescuing activity is a hormone synthesized by DAF-9 that acts through DAF-12.     

Rescue of vasopressin V2 receptor mutants by chemical chaperones: specificity and mechanism

Because missense mutations in genetic diseases of membrane proteins often result in endoplasmic reticulum (ER) retention of functional proteins, drug-induced rescue of their cell surface expression and understanding the underlying mechanism are of clinical value. To study this, we tested chemical chaperones and sarco(endo)plasmic reticulum Ca2+ ATPase pump inhibitors on Madin-Darby canine kidney cells expressing nine ER-retained vasopressin type-2 receptor (V2R) mutants involved in nephrogenic diabetes insipidus. Of these nine, only V2R-V206D showed improved maturation and plasma membrane rescue with glycerol, dimethyl sulfoxide (DMSO), thapsigargin/curcumin, and ionomycin but not with other osmolytes or growth at 27 degrees C. This revealed that rescue is mutant specific and that this mutant is prone to rescue by multiple compounds. Rescue did not involve changed expression of molecular chaperones calnexin, heat-shock protein (HSP) 70, or HSP90. V2R antagonist SR121463B treatment revealed that V2R-V206D and V2R-S167T were rescued and matured to a greater extent, suggesting that the rescuing activity of a pharmacological versus chemical chaperone is broader and stronger. Calcium measurements showed that rescue of V2R-V206D by thapsigargin, curcumin, and ionomycin was because of increased cytosolic calcium level, rather than decreased endoplasmic reticulum calcium level. The molecular mechanism underlying rescue by DMSO, glycerol, and SR121463B is different, because with these compounds intracellular calcium levels were unaffected.     

Characterization of the role of the FluG protein in asexual development of Aspergillus nidulans.

We showed previously that a DeltafluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG ( approximately 400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of DeltafluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from DeltaflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.     

Asexual sporulation signalling regulates autolysis of Aspergillus nidulans via modulating the chitinase ChiB production

Aims:  Elucidation of the regulation of ChiB production in Aspergillus nidulans .
Methods and Results:  Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental and/or autolytic mutants. Investigation of disorganization of mycelial pellets, loss of biomass, extra-/intracellular chitinase activities, ChiB production and chiB mRNA levels in various cultures revealed that, in submerged cultures, initialization of autolysis and stationary phase-induced ChiB production are intimately coupled, and that both processes are controlled by the FluG-BrlA asexual sporulation regulatory pathway. ChiB production does not affect the progression of apoptotic cell death in the aging A. nidulans cultures.
Conclusions:  The endochitinase ChiB plays an important role in autolysis of A. nidulans , and its production is initiated by FluG-BrlA signalling. Despite the fact that apoptosis is an inseparable part of fungal autolysis, its regulation is independent to FluG-initiated sporulation signalling.
Significance and Impact of the Study:  Deletion of chiB and fluG homologues in industrial filamentous fungal strains may stabilize the hyphal structures in the autolytic phase of growth and limit the release of autolytic hydrolases into the culture medium.     

Developmental defects resulting from arginine auxotrophy in Aspergillus nidulans

A mutant of Aspergillus nidulans, isolated for inability to form asexual spores (conidia) on complete medium, was found to regain the ability to conidiate if the medium was supplemented with arginine. On minimal medium the mutant required arginine for growth but at a much lower concentration than that required for conidiation. This mutant, designated argB12, thus defines a phase-critical gene, i.e. a gene whose function is in greater demand for development than for growth. In addition to its aconidial phenotype, the mutant also exhibited (depending on the medium) aberrant sexual development and a low efficiency of conidial germination. In crosses, each of these developmental phenotypes segregated with arginine auxotrophy. Genetic and biochemical analyses showed the argB12 mutation to be an allele of the previously described argB locus, mutants of which lack ornithine transcarbamylase. Arginine-requiring mutants at at least two other loci were also found to be defective in asexual sporulation, but the germination defect appears to be specific to argB mutants.     

Conditional Mutants of Meiosis in Yeast

Three temperature-sensitive mutants, spo1-1, spo2-1, and spo3-1, were characterized with respect to their behavior in sporulation medium at a restrictive temperature. The time of expression of the functions defective in the mutants was determined by temperature-shift experiments during the sporulation process. In addition, each mutant was examined for the following: (i) its ability to undergo the nuclear divisions of meiosis; (ii) deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis; (iii) protein turnover; and (iv) colony-forming ability after exposure to sporulation medium. Mutant spo1-1 is defective in a function which confers a temperature-sensitive period which extends over 32% of the sporulation cycle. The temperature-sensitive period of mutant spo2-1 occupies 34% of the cycle, whereas the temperature-sensitive period of mutant spo3-1 extends over 2% of the sporulation cycle. Cytological evidence indicates that all three mutants initiate but do not complete the meiotic nuclear divisions. The DNA content of sporulation cultures of mutants spo1-1 and spo3-1 did not increase to the wild-type level; DNA synthesis in spo2-1 was normal. All three strains exhibit a loss of colony-forming ability during incubation in sporulation medium at the restrictive temperature. RNA and protein synthesis and protein turnover occur in the mutants.     

Transgenically ectopic expression of Bmp4 to the Msx1 mutant dental mesenchyme restores downstream gene expression but represses Shh and Bmp2 in the enamel knot of wild type tooth germ

Bmp4 is a downstream gene of Msx1 in early mouse tooth development. In this study, we introduced the Msx1-Bmp4 transgenic allele to the Msx1 mutants in which tooth development is arrested at the bud stage in an effort of rescuing Msx1 mutant tooth phenotype in vivo. Ectopic expression of a Bmp4 transgene driven by the mouse Msx1promoter in the dental mesenchyme restored the expression of Lef-1 and Dlx2 but neither Fgf3 nor syndecan-1 in the Msx1 mutant molar tooth germ. The mutant phenotype of molar but not incisor could be partially rescued to progress to the cap stage. The Msx1-Bmp4 transgene was also able to rescue the alveolar processes and the neonatal lethality of the Msx1 mutants. In contrast, overexpression of Bmp4 in the wild type molar mesenchyme down-regulated Shh and Bmp2 expression in the enamel knot, the putative signaling center for tooth patterning, but did not produce a tooth phenotype. These results indicate that Bmp4 can bypass Msx1 function to partially rescue molar tooth development in vivo, and to support alveolar process formation. Expression of Shh and Bmp2 in the enamel knot may not represent critical signals for tooth patterning.