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Global transcriptome analysis of spore formation in Myxococcus xanthus reveals a locus necessary for cell differentiation.

Müller FD, Treuner-Lange A, Heider J, Huntley SM, Higgs PI - BMC Genomics (2010)

Bottom Line: Most of the previously identified sporulation marker genes were significantly upregulated.Furthermore, during the starvation-induced developmental program, these genes were expressed in fruiting bodies but not in peripheral rods, a subpopulation of developing cells which do not sporulate.These results suggest that microarray analysis of chemical-induced spore formation is an excellent system to specifically identify genes necessary for the core sporulation process of a Gram negative model organism for differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, 35043, Marburg, Germany.

ABSTRACT

Background: Myxococcus xanthus is a Gram negative bacterium that can differentiate into metabolically quiescent, environmentally resistant spores. Little is known about the mechanisms involved in differentiation in part because sporulation is normally initiated at the culmination of a complex starvation-induced developmental program and only inside multicellular fruiting bodies. To obtain a broad overview of the sporulation process and to identify novel genes necessary for differentiation, we instead performed global transcriptome analysis of an artificial chemically-induced sporulation process in which addition of glycerol to vegetatively growing liquid cultures of M. xanthus leads to rapid and synchronized differentiation of nearly all cells into myxospore-like entities.

Results: Our analyses identified 1 486 genes whose expression was significantly regulated at least two-fold within four hours of chemical-induced differentiation. Most of the previously identified sporulation marker genes were significantly upregulated. In contrast, most genes that are required to build starvation-induced multicellular fruiting bodies, but which are not required for sporulation per se, were not significantly regulated in our analysis. Analysis of functional gene categories significantly over-represented in the regulated genes, suggested large rearrangements in core metabolic pathways, and in genes involved in protein synthesis and fate. We used the microarray data to identify a novel operon of eight genes that, when mutated, rendered cells unable to produce viable chemical- or starvation-induced spores. Importantly, these mutants displayed no defects in building fruiting bodies, suggesting these genes are necessary for the core sporulation process. Furthermore, during the starvation-induced developmental program, these genes were expressed in fruiting bodies but not in peripheral rods, a subpopulation of developing cells which do not sporulate.

Conclusions: These results suggest that microarray analysis of chemical-induced spore formation is an excellent system to specifically identify genes necessary for the core sporulation process of a Gram negative model organism for differentiation.

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Related in: MedlinePlus

The nfs locus is necessary for formation of viable starvation-induced spores. A. Starvation induced developmental phenotype of the wild type (DK1622) and Δ(nfsA-H) (PH1200) strains. 4 × 107 cells were spotted onto nutrient limited CF agar plates, incubated at 32°C, and development was recorded at the indicated times. By 24 hours development cells aggregate into mounds (fruiting bodies) of approximately 105 cells. Fruiting bodies are displayed at higher magnification at 72 hours to illustrate that the Δ(nfsA-H) fruiting bodies fail to darken. B. Heat and sonication resistant spores (wild type; DK1622) and entities [Δ(nfsA-H); PH1200] isolated after 120 hours of development from A and examined by phase contrast microscopy.
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Figure 4: The nfs locus is necessary for formation of viable starvation-induced spores. A. Starvation induced developmental phenotype of the wild type (DK1622) and Δ(nfsA-H) (PH1200) strains. 4 × 107 cells were spotted onto nutrient limited CF agar plates, incubated at 32°C, and development was recorded at the indicated times. By 24 hours development cells aggregate into mounds (fruiting bodies) of approximately 105 cells. Fruiting bodies are displayed at higher magnification at 72 hours to illustrate that the Δ(nfsA-H) fruiting bodies fail to darken. B. Heat and sonication resistant spores (wild type; DK1622) and entities [Δ(nfsA-H); PH1200] isolated after 120 hours of development from A and examined by phase contrast microscopy.

Mentions: To determine whether the deletion mutant is also perturbed in starvation induced sporulation, the mutant was induced to develop on nutrient-limited CF agar plates. The mutant began to aggregate exactly as wild type and formed similar looking aggregates from 0-24 hours (Figure 4A). However, by 48 hours, the wild type fruiting bodies began to darken with the onset of sporulation, whereas the mutant fruiting bodies failed to darken even after continued incubation for 120 hours (Figure 4 and data not shown). After 120 hours, harvested cells were subject to heat and sonic disruption, and enumerated using a hemacytometer. While the wild type produced 2.2 ± 0.3 × 107 heat and sonication resistant entities, the mutant produced 6.3 ± 1 × 106 entities (28 ± 16% of wild type) which, when examined by phase contrast light microscopy, were non-refractile, slightly misshapen spheres compared to the spherical refractile wild type spores (Figure 4B). Germination ability was assayed by plating both wild type and mutant on nutrient rich agar, the mutant produced 5 ± 34% of wild type germinating spores.


Global transcriptome analysis of spore formation in Myxococcus xanthus reveals a locus necessary for cell differentiation.

Müller FD, Treuner-Lange A, Heider J, Huntley SM, Higgs PI - BMC Genomics (2010)

The nfs locus is necessary for formation of viable starvation-induced spores. A. Starvation induced developmental phenotype of the wild type (DK1622) and Δ(nfsA-H) (PH1200) strains. 4 × 107 cells were spotted onto nutrient limited CF agar plates, incubated at 32°C, and development was recorded at the indicated times. By 24 hours development cells aggregate into mounds (fruiting bodies) of approximately 105 cells. Fruiting bodies are displayed at higher magnification at 72 hours to illustrate that the Δ(nfsA-H) fruiting bodies fail to darken. B. Heat and sonication resistant spores (wild type; DK1622) and entities [Δ(nfsA-H); PH1200] isolated after 120 hours of development from A and examined by phase contrast microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875238&req=5

Figure 4: The nfs locus is necessary for formation of viable starvation-induced spores. A. Starvation induced developmental phenotype of the wild type (DK1622) and Δ(nfsA-H) (PH1200) strains. 4 × 107 cells were spotted onto nutrient limited CF agar plates, incubated at 32°C, and development was recorded at the indicated times. By 24 hours development cells aggregate into mounds (fruiting bodies) of approximately 105 cells. Fruiting bodies are displayed at higher magnification at 72 hours to illustrate that the Δ(nfsA-H) fruiting bodies fail to darken. B. Heat and sonication resistant spores (wild type; DK1622) and entities [Δ(nfsA-H); PH1200] isolated after 120 hours of development from A and examined by phase contrast microscopy.
Mentions: To determine whether the deletion mutant is also perturbed in starvation induced sporulation, the mutant was induced to develop on nutrient-limited CF agar plates. The mutant began to aggregate exactly as wild type and formed similar looking aggregates from 0-24 hours (Figure 4A). However, by 48 hours, the wild type fruiting bodies began to darken with the onset of sporulation, whereas the mutant fruiting bodies failed to darken even after continued incubation for 120 hours (Figure 4 and data not shown). After 120 hours, harvested cells were subject to heat and sonic disruption, and enumerated using a hemacytometer. While the wild type produced 2.2 ± 0.3 × 107 heat and sonication resistant entities, the mutant produced 6.3 ± 1 × 106 entities (28 ± 16% of wild type) which, when examined by phase contrast light microscopy, were non-refractile, slightly misshapen spheres compared to the spherical refractile wild type spores (Figure 4B). Germination ability was assayed by plating both wild type and mutant on nutrient rich agar, the mutant produced 5 ± 34% of wild type germinating spores.

Bottom Line: Most of the previously identified sporulation marker genes were significantly upregulated.Furthermore, during the starvation-induced developmental program, these genes were expressed in fruiting bodies but not in peripheral rods, a subpopulation of developing cells which do not sporulate.These results suggest that microarray analysis of chemical-induced spore formation is an excellent system to specifically identify genes necessary for the core sporulation process of a Gram negative model organism for differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, 35043, Marburg, Germany.

ABSTRACT

Background: Myxococcus xanthus is a Gram negative bacterium that can differentiate into metabolically quiescent, environmentally resistant spores. Little is known about the mechanisms involved in differentiation in part because sporulation is normally initiated at the culmination of a complex starvation-induced developmental program and only inside multicellular fruiting bodies. To obtain a broad overview of the sporulation process and to identify novel genes necessary for differentiation, we instead performed global transcriptome analysis of an artificial chemically-induced sporulation process in which addition of glycerol to vegetatively growing liquid cultures of M. xanthus leads to rapid and synchronized differentiation of nearly all cells into myxospore-like entities.

Results: Our analyses identified 1 486 genes whose expression was significantly regulated at least two-fold within four hours of chemical-induced differentiation. Most of the previously identified sporulation marker genes were significantly upregulated. In contrast, most genes that are required to build starvation-induced multicellular fruiting bodies, but which are not required for sporulation per se, were not significantly regulated in our analysis. Analysis of functional gene categories significantly over-represented in the regulated genes, suggested large rearrangements in core metabolic pathways, and in genes involved in protein synthesis and fate. We used the microarray data to identify a novel operon of eight genes that, when mutated, rendered cells unable to produce viable chemical- or starvation-induced spores. Importantly, these mutants displayed no defects in building fruiting bodies, suggesting these genes are necessary for the core sporulation process. Furthermore, during the starvation-induced developmental program, these genes were expressed in fruiting bodies but not in peripheral rods, a subpopulation of developing cells which do not sporulate.

Conclusions: These results suggest that microarray analysis of chemical-induced spore formation is an excellent system to specifically identify genes necessary for the core sporulation process of a Gram negative model organism for differentiation.

Show MeSH
Related in: MedlinePlus