The asg mutants of Myxococcus xanthus fail to produce a set of related substances called A-factor. A-factor is released into the medium and is required early in fruiting body development. Lacking A-factor, the asg mutants are defective in aggregation, sporulation, and expression of most genes whose products appear later than 1 h after development is induced by starvation. Previous work has shown that these defects are reversed when A-factor, released by developing wild-type cells, is added to asg mutant cells. Part of the material in conditioned medium with A-factor activity is heat stable and dialyzable. This low-molecular-weight A-factor consists of a mixture of amino acids and peptides. Fifteen single amino acids have A-factor activity, and 11 of these are found in conditioned medium. Mixtures of amino acids have a total activity approximately equal to the sum of the activities of their constituents. Conditioned medium also contains peptides with A-factor activity. Pure peptides have A-factor activity, and their specific activities are equal to or less than the sum of the activities of their constituent amino acids. There is no evidence for a specialized A-factor peptide in conditioned medium, one with a specific activity greater than the sum of its constituent amino acids. About half of the heat-stable A-factor activity in conditioned medium can be accounted for by free amino acids, and the remaining half can be accounted for by peptides. It is argued that heat-stable A-factor induces A-dependent gene expression not by the nutritional action of amino acids but through a chemosensory circuit.
Mutations in any of three asg (A-signalling) loci cause fruiting body development of Myxococcus xanthus to arrest at about the 2-h stage. Development can be restored to asg mutants by the addition of conditioned buffer in which wild-type cells have been developing or of A-factor purified from the conditioned buffer. Two forms of A-factor have been identified: heat-stable A-factor, which is composed of amino acids and peptides, and heat-labile A-factor, which consists of at least two proteases. A-factor is found in conditioned buffer in rough proportion to the cell density. As decreasing amounts of either form of A-factor are added, the developmental response of asg cells decreases until a threshold concentration is reached, below which no response is detected. In addition, wild-type cells fail to develop when their density is decreased below the point at which the level of A-factor is predicted to fall short of this threshold. The development of low-density asg+ cells can, however, be restored by the addition of either form of A-factor. These experiments show that A-factor is important for the development of wild-type cells. Moreover, the development of an asgB mutant that produces 5 to 10% the wild-type level of A-factor can be restored when the cell density is increased 10-fold above the standard density. We propose that the A-signal is used by M. xanthus to specify the minimum cell density required for the initiation of development. Differences in the response to A-factor between different asg mutants suggest that the different asg loci govern A-factor production in diverse ways.
The asg mutants of Myxococcus xanthus are defective in the production of an extracellular substance, called A-factor, that is required for expression of a set of fruiting body-specific genes. A-factor is released by wild-type cells (asg+) after 1 to 2 h of development. When A-factor is added to asg mutant cells, it restores expression of their A-factor-dependent genes. Rescue of beta-galactosidase production in an asg mutant carrying the A-factor-dependent lacZ transcriptional fusion (omega 4521) was used to assay A-factor activity. According to this assay, two types of substances with A-factor activity are present in conditioned medium. One type is heat stable and of low molecular weight; the other is heat labile and of high molecular weight. An approximately 27-kDa protein with heat-labile A-factor activity was purified from conditioned medium. The purified protein has proteolytic activity as well as A-factor activity. The substrate specificity of the 27-kDa protease resembles that of trypsin. A smaller protein with both heat-labile A-factor activity and proteolytic activity was identified. Its substrate specificity differs from that of the 27-kDa protein. In addition, trypsin and other proteases were found to have heat-labile A-factor activity. Trypsin inhibitory protein from soybeans neutralizes the A-factor activity of trypsin in parallel with its neutralization of protease activity, showing that the proteolytic activity of trypsin is necessary for its A-factor activity. The 27-kDa protein rescues the aggregation and sporulation defects of an asgB mutant in submerged culture as well as its ability to express beta-galactosidase from an asg-dependent lac fusion.
The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro.
We isolated an Escherichia coli methionine auxotroph that displays a growth phenotype similar to that of known metF mutants but has elevated levels of 5,10-methylenetetrahydrofolate reductase, the metF gene product. Transduction analysis indicates that (i) the mutant carries normal metE, metH, and metF genes; (ii) the phenotype is due to a single mutation, eliminating the possibility that the strain is a metE metH double mutant; and (iii) the new mutation is linked to the metE gene by P1 transduction. Plasmids carrying the Salmonella typhimurium metE gene and flanking regions complement the mutation, even when the plasmidborne metE gene is inactivated. Enzyme assays show that the mutation results in a dramatic decrease in metE gene expression, a moderate decrease in metH gene expression, and a disruption of the metH-mediated vitamin B12 repression of the metE and metF genes. Our evidence suggests that the methionine auxotrophy caused by the new mutation is a result of insufficient production of both the vitamin B12-independent (metE) and vitamin B12-dependent (metH) transmethylase enzymes that are necessary for the synthesis of methionine from homocysteine. We propose that this mutation defines a positive regulatory gene, designated metR, whose product acts in trans to activate the metE and metH genes.The methylation of homocysteine to form methionine can be carried out by either of two transmethylases in Salmonella typhimurium and Escherichia coli (for a review, see reference 15). The first is a vitamin B12-independent enzyme, the product of the metE gene; the second is a vitamin B12-dependent enzyme, the product of the metH gene. The methyl donor for both enzymes is 5-methyltetrahydrofolate, produced by the metF gene product at a point of convergence of two major pathways, the methionine biosynthetic pathway and the C1 pathway (Fig. 1). The cell regulates the flow of C1 units through this convergence point on several levels to balance the requirements for protein synthesis, methylation reactions, and nucleic acid synthesis.The genes in the nonfolate branch of the methionine pathway (metA, metB, metC, and metK) and those in the folate branch of the pathway (metF, metE, and, to a small extent, metH) are all negatively controlled by the metJ repressor system. In addition, the metH gene product is involved in repression of the metE and metF genes when the cells are grown in medium containing vitamin B12. We report here the finding of a third regulatory mechanism at the methionine-C, convergence point, namely, the positive activation of the metE and metH genes. MATERIALS AND METHODSBacterial strains, plasmids, and bacteriophages. All bacterial strains used are derivatives of E. coli K-12 and are described in Table 1. Plasmids pGS47 and pGS69, and their metE::Tn5 derivatives have been described previously (16). Plasmid pMC1403 (4) was from M. Casadaban. Bacteriophage Xgt2 (13) was from R. Davis. Plasmid pBR322 has been described previously (9). Plasmid pGS191, the lacZ * Corresponding author. fusion plasmids, and la...
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