Role of autocide AMI in development of Myxococcus xanthus.

A new developmental mutant of Myxococcus xanthus has been isolated by screening TnV insertion mutants for AMI-dependent development in submerged culture. This mutant (ER304) aggregated and sporulated on agar surfaces but required at least 3.8 micrograms of autocide AMI per ml for development in submerged cultures. Spore rescue of ER304 was obtained with the saturated, monounsaturated, and diunsaturated fatty acid fractions of AMI, with specific activities of 68, 115, and 700 U/mg, respectively. In addition, several model fatty acids were capable of rescuing sporulation of ER304; however, there was no correlation between specific lytic activity observed in vegetative cultures and specific rescue activity. Rescue of ER304 was effected during the first ca. 12 h after the initiation of starvation conditions; after this time, addition of AMI or model fatty acids killed the cells. Supernatant fluids of ER304 rescued development in dsg mutants (e.g., DK3260) in submerged cultures, but dsg mutant supernatant fluids were incapable of rescuing ER304 development. The data presented in this article support the idea that the primary mechanism of rescue by AMI is not via lysis, although developmental lysis may be an indirect result of the rescue event. A membrane permeability model is presented to explain the role of autocides in early developmental events in wild-type strains and in the aggregation and sporulation rescue of developmental mutants ER304 and DK3260.     

Identification of the RNA products of the ops gene of Myxococcus xanthus and mapping of ops and tps RNAs.

The expression of the ops gene, like that of the highly homologous and closely linked tps gene, is induced during development of the fruiting bacterium Myxococcus xanthus. The RNA products of the ops gene have been identified and compared with tps RNA. The ops RNA was observed in developmental cells only after spore formation had commenced, and it was necessary to use a sporulation-defective mutant strain or to disrupt spores to isolate this RNA. RNA from the ops gene was not observed in vegetative cells but was readily detected in cells subjected to glycerol-induced sporulation. In contrast, a large amount of developmental tps RNA was observed in cells well before sporulation had occurred; low levels of tps RNA were observed in vegetative cells; and only a slight increase in tps RNA was found during glycerol-induced sporulation. Several ops and tps RNAs were observed in this study, and the positions of these RNAs were mapped on the M. xanthus genome. The 5' ends of both the ops and tps RNAs mapped predominantly to positions about 50 bases upstream from the respective translational initiation sites. The 3' ends of RNAs from both genes were heterogeneous. The four ops RNAs were 620, 775, 845, and 1,230 bases in length, while the tps RNAs were 612, 695, 730, and 935 bases.     

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.     

An effective sporulation of Myxococcus xanthus requires glycogen consumption via Pkn4-activated 6-phosphofructokinase

6-Phosphofructokinase (PFK) is a key enzyme for glycolysis in both prokaryotes and eukaryotes. Previously, it was found that the activity of Myxococcus xanthus PFK increased 2.7-fold upon phosphorylation at Thr-226 by the Ser/Thr kinase Pkn4. The pkn4 gene is located 18 bp downstream of the pfk gene forming an operon, and both genes are expressed during vegetative growth and development. Here, we show that glycogen, which accumulates during stationary phase and early in development, is consumed during sporulation. A pfk-pkn4 deletion strain accumulated glycogen at a higher level than the wild-type strain, was unable to consume glycogen during developmental progression and exhibited a poor spore yield. From genetic complementation analysis of the pfk-pkn4 deletion strain with the pfk and pkn4 genes, it was found that glycogen consumption and a high spore yield require not only the pfk gene but also the pkn4 gene. Furthermore, phosphorylation is critical for glycogen consumption because the pfk gene engineered to express the mutant PFK (Thr-226-Ala) did not complement a pfk mutant. We propose that glycogen metabolism in M. xanthus is regulated in a similar manner to that in eukaryotes requiring a protein Ser/Thr kinase.     

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.     

Mutational analysis of the Myxococcus xanthus Omicron4499 promoter region reveals shared and unique properties in comparison with other C-signal-dependent promoters

The bacterium Myxococcus xanthus undergoes multicellular development during times of nutritional stress and uses extracellular signals to coordinate cell behavior. C-signal affects gene expression late in development, including that of Omega4499, an operon identified by insertion of Tn5 lac into the M. xanthus chromosome. The Omega4499 promoter region has several sequences in common with those found previously to be important for expression of other C-signal-dependent promoters. To determine if these sequences are important for Omega4499 promoter activity, the effects of mutations on expression of a downstream reporter gene were tested in M. xanthus. Although the promoter resembles those recognized by Escherichia coli sigma(54), mutational analysis implied that a sigma(70)-type sigma factor likely recognizes the promoter. A 7-bp sequence known as a C box and a 5-bp element located 6 bp upstream of the C box have been shown to be important for expression of other C-signal-dependent promoters. The Omega4499 promoter region has C boxes centered at -33 and -55 bp, with 5-bp elements located 7 and 8 bp upstream, respectively. A multiple-base-pair mutation in any of these sequences reduced Omega4499 promoter activity more than twofold. Single base-pair mutations in the C box centered at -33 bp yielded a different pattern of effects on expression than similar mutations in other C boxes, indicating that each functions somewhat differently. An element from about -81 to -77 bp exerted a twofold positive effect on expression but did not appear to be responsible for the C-signal dependence of the Omega4499 promoter. Mutations in sigD and sigE, which are genes that encode sigma factors, reduced expression from the Omega4499 promoter. The results provide further insight into the regulation of C-signal-dependent genes, demonstrating both shared and unique properties among the promoter regions so far examined.     

Identification of major sporulation proteins of Myxococcus xanthus using a proteomic approach

Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress.     

A new putative sigma factor of Myxococcus xanthus.

A third putative sigma factor gene, sigC, has been isolated from Myxococcus xanthus by using the sigA gene (formerly rpoD of M. xanthus) as a probe. The nucleotide sequence of sigC has been determined, and an open reading frame of 295 residues (M(r) = 33,430) has been identified. The deduced amino acid sequence of sigC exhibits the features which are characteristic of other bacterial sigma factors. The characterization of a sigC-lacZ strain has demonstrated that sigC expression is induced immediately after cells enter into the developmental cycle and is dramatically reduced at the onset of sporulation. A deletion mutant of sigC grows normally in vegetative culture and is able to develop normally. However, in contrast to the wild-type cells, the sigC deletion mutant cells became capable of forming fruiting bodies and myxospores on semirich agar plates. This suggests that sigC may play a role in expression of genes involved in negatively regulating the initiation of fruiting body formation.     

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.     

Propionyl coenzyme A carboxylase is required for development of Myxococcus xanthus.

A dcm-1 mutant, obtained by transposon mutagenesis of Myxococcus xanthus, could aggregate and form mounds but was unable to sporulate under nutrient starvation. A sequence analysis of the site of insertion of the transposon showed that the insertion lies within the 3' end of a 1,572-bp open reading frame (ORF) designated the M. xanthus pccB ORF. The wild-type form of the M. xanthus pccB gene, obtained from a lambdaEMBL library of M. xanthus, shows extensive similarity to a beta subunit of propionyl coenzyme A (CoA) carboxylase, an alpha subunit of methylmalonyl-CoA decarboxylase, and a 12S subunit of transcarboxylase. In enzyme assays, extracts of the dcm-1 mutant were deficient in propionyl-CoA carboxylase activity. This enzyme catalyzes the ATP-dependent carboxylation of propionyl-CoA to yield methylmalonyl-CoA. The methylmalonyl-CoA rescued the dcm-1 mutant fruiting body and spore development. During development, the dcm-1 mutant cells also had reduced levels of long-chain fatty acids (C16 to C18) compared to wild-type cells.