Identification and characterization of genes required for early Myxococcus xanthus developmental gene expression

Starvation and cell density regulate the developmental expression of Myxococcus xanthus gene 4521. Three classes of mutants allow expression of this developmental gene during growth on nutrient agar, such that colonies of strains containing a Tn5 lac Omega4521 fusion are Lac(+). One class of these mutants inactivates SasN, a negative regulator of 4521 expression; another class activates SasS, a sensor kinase-positive regulator of 4521 expression; and a third class blocks lipopolysaccharide (LPS) O-antigen biosynthesis. To identify additional positive regulators of 4521 expression, 11 Lac(-) TnV.AS transposon insertion mutants were isolated from a screen of 18,000 Lac(+) LPS O-antigen mutants containing Tn5 lac Omega4521 (Tc(r)). Ten mutations identified genes that could encode positive regulators of 4521 developmental expression based on their ability to abolish 4521 expression during development in the absence of LPS O antigen and in an otherwise wild-type background. Eight of these mutations mapped to the sasB locus, which encodes the known 4521 regulators SasS and SasN. One mapped to sasS, whereas seven identified new genes. Three mutations mapped to a gene encoding an NtrC-like response regulator homologue, designated sasR, and four others mapped to a gene designated sasP. One mutation, designated ssp10, specifically suppressed the LPS O-antigen defect; the ssp10 mutation had no effect on 4521 expression in an otherwise wild-type background but reduced 4521 developmental expression in the absence of LPS O antigen to a level close to that of the parent strain. All of the mutations except those in sasP conferred defects during growth and development. These data indicate that a number of elements are required for 4521 developmental expression and that most of these are necessary for normal growth and fruiting body development.     

Identification and characterization of the Myxococcus xanthus bsgA gene product.

The bsgA mutants of Myxococcus xanthus are blocked at a very early stage of the developmental program. They fail to produce fruiting bodies or to sporulate under normal conditions but can be rescued by extracellular complementation in mixtures with wild-type cells. A bsgA-lacZ gene fusion was constructed and expressed in Escherichia coli. The resulting fusion protein, which has beta-galactosidase enzyme activity, was partially purified by affinity chromatography and preparative polyacrylamide gel electrophoresis. The protein was used to immunize mice, which produced a hybridoma secreting monoclonal antibody that was specific for the bsgA gene product. The monoclonal antibody was used in Western blot (immunoblot) experiments to determine the apparent cellular location of the bsgA protein in M. xanthus and to compare the level of this protein at various times in the Myxococcus life cycle.     

Suppressors that permit A-signal-independent developmental gene expression in Myxococcus xanthus.

Progression through the early stages of Myxococcus xanthus fruiting body development requires the cell-to-cell transmission of soluble material called A signal. During these early stages, expression from the gene identified by Tn5 lac insertion omega 4521 increases. A DNA probe of the omega 4521 gene was constructed. Use of this probe showed that accumulation of mRNA corresponding to the omega 4521 gene depends upon A signal. A-signal-deficient (asg) mutants fail to accumulate this RNA, and the external addition of A signal restores accumulation. To identify links between A signal and its responsive gene, omega 4521, suppressors of an asg mutation were generated. All of the suppressor alleles restored lacZ expression from omega 4521 in the absence of A signal, and they were demonstrated to be neither reversions of the asgB mutation nor mutations in the promoter of omega 4521. Fifteen suppressor mutations map to two loci, sasA and sasB (for suppressor of asg). sasA and sasB mutants differ phenotypically during growth and development. Mid-logarithmic-phase sasA asgB double mutants, like sas+ asg+ strains, express low levels of lacZ, whereas sasB asgB double mutants express high levels. sasA asg+ mutants form abnormal colonies, are less cohesive than wild type, and are defective in fruiting body formation and sporulation. In contrast, sasB asg+ mutants form normal colonies, are as cohesive as wild type, and appear to develop normally. The characteristics of sasA suppressors implicate the sasA+ product as a negative regulator in the A-signal-dependent regulation of omega 4521.     

Identification of a putative eukaryotic-like protein kinase family in the developmental bacterium Myxococcus xanthus.

Myxococcus xanthus is a gram-negative bacterium which, upon starvation, undergoes a spectacular developmental cycle culminating in the formation of spore-filled fruiting bodies. We recently characterized a protein serine-threonine kinase (Pkn1) that is required for normal development (J. Munoz-Dorado, S. Inouye, and M. Inouye, Cell 67:995-1006, 1991). pkn1 was cloned by polymerase chain reaction amplification with primers designed from conserved sequences in eukaryotic protein kinases. In this study, a fragment of the pkn1 gene and an oligonucleotide corresponding to another highly conserved region were employed as probes for Southern blot analyses, which indicated that there are at least 26 putative kinase genes in M. xanthus. Most of the putative kinase genes were cloned, and complete or partial sequencing of eight clones revealed that they indeed contained highly conserved sequences present in eukaryotic kinases. These results suggest that complex kinase cascades similar to those described for eukaryotes might be involved in regulation of the M. xanthus life cycle.     

Control of developmental gene expression by cell-to-cell interactions in Myxococcus xanthus.

The ssbA mutants of Myxococcus xanthus behave as if they are unable to produce a cell-to-cell signal required for normal development. They are unable to form fruiting bodies or spores on developmental medium. They do sporulate, however, if allowed to develop in mixtures with wild-type cells. Fusions of developmentally induced promoters of M. xanthus to the Escherichia coli lacZ gene were used to characterize the effect of the ssbA mutations on developmental gene expression. Each of the five independent fusions tested was found to be dependent upon the ssbA+ allele for full expression. The ssbA mutants were able to express each of these fusions if the mutants were allowed to develop in mixtures with wild-type (Lac-) cells. These results cannot be explained on the basis of genetic exchange. The data are consistent with regulation of gene expression mediated by cell-to-cell interactions.     

Myxococcus xanthus sasS encodes a sensor histidine kinase required for early developmental gene expression.

Initiation of Myxococcus xanthus multicellular development requires integration of information concerning the cells' nutrient status and density. A gain-of-function mutation, sasB7, that bypasses both the starvation and high cell density requirements for developmental expression of the 4521 reporter gene, maps to the sasS gene. The wild-type sasS gene was cloned and sequenced. This gene is predicted to encode a sensor histidine protein kinase that appears to be a key element in the transduction of starvation and cell density inputs. The sasS null mutants express 4521 at a basal level, form defective fruiting bodies, and exhibit reduced sporulation efficiencies. These data indicate that the wild-type sasS gene product functions as a positive regulator of 4521 expression and participates in M. xanthus development. The N terminus of SasS is predicted to contain two transmembrane domains that would locate the protein to the cytoplasmic membrane. The sasB7 mutation, an E139K missense mutation, maps to the predicted N-terminal periplasmic region. The C terminus of SasS contains all of the conserved residues typical of the sensor histidine protein kinases. SasS is predicted to be the sensor protein in a two-component system that integrates information required for M. xanthus developmental gene expression.     

Intercellular signaling is required for developmental gene expression in Myxococcus xanthus

Certain developmental mutants of Myxococcus xanthus can be complemented (extracellularly) by wild-type cells. Insertions of Tn5 lac (a transposon which couples beta-galactosidase expression to exogenous promoters) into developmentally regulated genes were used to investigate extracellular complementation of the A group mutations. A- mutations reduced developmental beta-galactosidase expression from 18 of 21 Tn5 lac insertions tested and that expression was restored to A- Tn5 lac cells by adding wild-type cells. The earliest A-dependent Tn5 lac normally expresses beta-galactosidase at 1.5 hr of development indicating a developmental block at 1-2 hr in A- mutants. A substance which can rescue the expression of this early Tn5 lac is released by wild-type (A+) but not by A- cells. This substance appears in a cell-free wash of wild-type cells or in starvation buffer conditioned by wild-type cells 1-2 hr after development is initiated. The conditioned starvation buffer also restores normal morphological development to an A- mutant.     

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.     

Site-specific integration and expression of a developmental promoter in Myxococcus xanthus.

A series of intercellular signals are involved in the regulation of gene expression during fruiting body formation of Myxococcus xanthus. Mutations which block cell interactions, such as csgA (formerly known as spoC), also prevent expression of certain developmentally regulated promoters. csgA+ cells containing Tn5 lac omega DK4435, a developmentally regulated promoter fused to lacZ, began synthesizing lacZ mRNA 12 to 18 h into the developmental cycle. beta-Galactosidase specific activity increased about 12 h later. Neither lacZ mRNA nor beta-galactosidase activity was detected in a developing csgA mutant containing omega DK4435. The developmental promoter and its fused lacZ reporter gene were cloned into a pBR322-derived plasmid vector containing a portion of bacteriophage Mx8. These plasmids preferentially integrated into the M. xanthus chromosome by site-specific recombination at the bacteriophage Mx8 attachment site and maintained a copy number of 1 per chromosome. The integrated plasmids were relatively stable, segregating at a frequency of 0.0007% per generation in the absence of selection. The cloned and integrated promoter behaved like the native promoter, expressing beta-galactosidase at the proper time during wild-type development and failing to express the enzyme during development of a csgA mutant. The overall level of beta-galactosidase expression in merodiploid cells containing one native promoter and one promoter fused to lacZ was about half that of cells containing a single promoter fused to lacZ. These results suggest that the timing of developmentally regulated gene expression is largely independent of the location of this gene within the chromosome. Furthermore, they show that site-specific recombination can be a useful tool for establishing assays for promoter or gene function in M. xanthus.     

Genetic characterization of aggregation-defective developmental mutants of Myxococcus xanthus.

The transposon Tn5 was used to map temperature-sensitive mutants of Myxococcus xanthus defective in aggregation (C. E. Morrison and D. R. Zusman, J. Bacteriol. 140:1036-1042, 1979). Seven of the eight mutants showing a similar terminal phenotype (rough) were found to be tightly linked. These mapped in a group of loci which we have designated aggR1, aggR2, aggR3, and aggR4. Temperature-sensitive mutants having a different terminal phenotype were not liked to aggR. A search through a group of nonconditional rough mutants indicated that a much lower proportion of these (1 of 35) mapped in aggR. Thus, aggR is probably only one of many sites which can lead to the rough phenotype when mutated. Localized mutagenesis was used to isolate nine additional aggR mutants. All mapped within aggR1, aggR2, or aggR3, and none was found outside this region. Thus, we have characterized a cluster of developmental genes which are needed for aggregation in M. xanthus. The localization of a Tn5 insert adjacent to this region makes possible further manipulation of these genes.     

Murein components rescue developmental sporulation of Myxococcus xanthus

Murein (peptidoglycan) components are able to rescue sporulation in certain sporulation-defective mutants of Myxococcus xanthus. N-Acetylglucosamine, N-acetylmuramic acid, diaminopimelic acid, and D-alanine each increase the number of spores produced by SpoC mutants. When all four components are included they have a synergistic effect, raising the number of spores produced by SpoC mutants to the wild-type level. Murein-rescued spores are resistant to heat and sonic oscillation and germinate when plated on a nutrient-rich medium. They appear to be identical to fruiting body spores in their ultrastructure, in their protein composition, and in their resistance to boiling sodium dodecyl sulfate. Murein rescue of sporulation, like fruiting body sporulation, requires high cell density, a low nutrient level, and a solid surface.     

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.     

Mutational analysis of the Myxococcus xanthus Omega4400 promoter region provides insight into developmental gene regulation by C signaling

Myxococcus xanthus utilizes extracellular signals during development to coordinate cell movement, differentiation, and changes in gene expression. One of these signals, the C signal, regulates the expression of many genes, including Omega4400, a gene identified by an insertion of Tn5 lac into the chromosome. Expression of Tn5 lac Omega4400 is reduced in csgA mutant cells, which fail to perform C signaling, and the promoter region has several sequences similar to sequences found in the regulatory regions of other C-signal-dependent genes. One such gene, Omega4403, depends absolutely on the C signal for expression, and its promoter region has been characterized previously by mutational analysis. To determine if the similar sequences within the Omega4400 and Omega4403 regulatory regions function in the same way, deletion analysis and site-directed mutagenesis of the Omega4400 promoter region were performed. A 7-bp sequence centered at -49 bp, termed a C box, is identical in the Omega4400 and Omega4403 promoter regions, yet mutations in the individual base pairs affected expression from the two promoters very differently. Also, a single-base-pair change within a similar 5-bp element, which is centered at -61 bp in both promoter regions, had very different effects on the activities of the two promoters. Further mutational analysis showed that two regions are important for Omega4400 expression; one region, from -63 to -31 bp, is required for Omega4400 expression, and the other, from -86 to -81 bp, exerts a two- to fourfold effect on expression and is at least partially responsible for the C signal dependence of the Omega4400 promoter. Mutations in sigD and sigE, which are genes that encode sigma factors, abolished and reduced Omega4400 expression, respectively. Expression of Omega4400 in actB or actC mutants correlated well with the altered levels of C signal produced in these mutants. Our results provide the first detailed analysis of an M. xanthus regulatory region that depends partially on C signaling for expression and indicate that similar DNA sequences in the Omega4400 and Omega4403 promoter regions function differently.