Biomedical subjects

L Plamann

Publications and source records attributed to L Plamann.

8 recordsLinked to original sources

PubMed1996

A Myxococcus xanthus cell density-sensing system required for multicellular development.

Progression through early Myxococcus xanthus multicellular fruiting body development requires the generation of and response to extracellular A signal. Extracellular A signal is a specific set of amino acids at an extracellular concentration greater than 10 muM. It functions as a cell density signal during starvation that allows the cells to sense that a minimal cell density has been reached and development can proceed. The generation of extracellular A signal requires the products of three asg genes. They have recently been identified as AsgA, a fused two-component histidine protein kinase and response regulator; AsgB, a putative DNA-binding protein; and AsgC, the M, xanthus major sigma factor. Other elements of the A signaling pathway map to the sasB locus and appear to be A signal transducers. These elements are regulators of the earliest A signal-dependent gene, whose promoter is a member of the sigma-54 family. Continued study of the A signaling pathway is expected to identify additional components of this network required for the complex behavioural response of fruiting body formation.

Cell Count↗

PubMed1996

Purification and in vitro phosphorylation of Myxococcus xanthus AsgA protein.

The deduced amino acid sequence of the Myxococcus xanthus AsgA protein contains an N-terminal domain that is homologous to the receiver of response regulators and a C-terminal domain that is homologous to the transmitter of histidine protein kinases. We overexpressed affinity-tagged AsgA in Escherichia coli, purified the recombinant protein, and showed that AsgA has autokinase activity in vitro. The results of chemical-stability assays suggest that AsgA is phosphorylated on a histidine and provide no evidence for transfer of the phosphoryl group to the conserved aspartate of the receiver domain.

Bacterial Proteins↗

PubMed1995

A missense mutation in rpoD results in an A-signalling defect in Myxococcus xanthus.

The Myxococcus xanthus asg genes (asgA, asgB, and asgC) are necessary for production of extracellular A-signal, which is thought to function as a cell-density signal. Previous analyses of the asgA and asgB genes suggest that they perform regulatory functions. In this work, we localized asgC to a region that contains genes homologous to rpsU, dnaG, and rpoD of the Escherichia coli macromolecular synthesis (MMS) operon. Surprisingly, asgC767 was found to be a mutant allele of rpoD, the gene encoding the major sigma factor of M. xanthus. The mutation in asgC767 results in a glutamate to lysine substitution at amino acid 598, which lies within conserved region 3.1 of the major sigma factors. Previous studies have shown that the asg mutants share a number of growth and developmental phenotypes. We found that A-signal restores developmental expression of an A-signal-dependent gene (omega 4521) in the asgC767 (rpoDEK598) mutant background in a manner similar to that seen in the asgA and asgB mutants. Because the asg mutants have very similar phenotypes and the asg genes encode proteins that appear to have regulatory functions, we hypothesize that the asg gene products function together in a regulatory pathway that is required for extracellular A-signal production.

Amino Acid Sequence↗

PubMed1995

The Myxococcus xanthus asgA gene encodes a novel signal transduction protein required for multicellular development.

The Myxococcus xanthus asgA gene is one of three known genes necessary for the production of extracellular A-signal, a cell density signal required early in fruiting body development. We determined the DNA sequence of asgA. The deduced 385-amino-acid sequence of AsgA was found to contain two domains: one homologous to the receiver domain of response regulators and the other homologous to the transmitter domain of histidine protein kinases. A kanamycin resistance (Kmr) gene was inserted at various positions within or near the asgA gene to determine the null phenotype. Those strains with the Kmr gene inserted upstream or downstream of asgA are able to form fruiting bodies, while strains containing the Kmr gene inserted within asgA fail to develop. The nature and location of the asgA476 mutation were determined. This mutation causes a leucine-to-proline substitution within a conserved stretch of hydrophobic residues in the N-terminal receiver domain. Cells containing the insertion within asgA and cells containing the asgA476 substitution have similar phenotypes with respect to development, colony color, and expression of an asg-dependent gene. An analysis of expression of a translational asgA-lacZ fusion confirms that asgA is expressed during growth and early development. Finally, we propose that AsgA functions within a signal transduction pathway that is required to sense starvation and to respond with the production of extracellular A-signal.

Amino Acid Sequence↗

PubMed1994

Evidence that asgB encodes a DNA-binding protein essential for growth and development of Myxococcus xanthus.

The asg mutants of Myxococcus xanthus are defective in production of extracellular A-signal, which serves as a cell density signal for fruiting-body development. The DNA sequence of asgB, one of the three asg genes, was determined. The deduced amino acid sequence of AsgB contains a DNA-binding helix-turn-helix motif near the C terminus. This putative helix-turn-helix is highly similar to the helix-turn-helix in region 4.2 of major sigma factors, which is the region that recognizes and interacts with -35 sequences of promoters. We propose that AsgB is a transcription factor that binds to DNA sequences similar to the -35 hexamer, TTGACA. Analyses of asgB RNA levels and expression of an asgB-lacZ translational fusion indicate that expression of asgB remains fairly constant during the transition from growth into early development. The mutation within the asgB480 allele was identified as an A-to-G transition that results in a threonine-to-alanine substitution in the predicted protein product. Attempts to replace the wild-type copy of asgB with a null allele failed, indicating that asgB may be essential for growth.

Amino Acid Sequence↗

PubMed1992

Proteins that rescue A-signal-defective mutants of Myxococcus xanthus.

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.

Amino Acid Sequence↗

PubMed1992

Identification of heat-stable A-factor from Myxococcus xanthus.

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.

Amino Acid Sequence↗

PubMed1992

A-signalling and the cell density requirement for Myxococcus xanthus development.

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.

4-Butyrolactone↗