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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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nfs genes are upregulated late during starvation induced development and expressed only within sporulating cells. A. The starvation-induced developmental program was induced in submerged culture for strains expressing mCherry fused after the promoter from pilA (PpilA::mCherry; strain PH1221), the 500 bp upstream of nfsA (PnfsA::mCherry; strain PH1220) or the vector backbone (vector; strain PH1222) in the DK1622 background. At the indicated times, mCherry fluorescence was recorded in a plate reader. B. Constructs expressing mCherry fused after the promoter from pilA (PpilA::mCherry; strain PH1221), the 500 bp upstream of nfsA (PnfsA::mCherry; strain PH1220) or the vector backbone (vector; strain PH1222) in the DK1622 background were induced to develop under submerged culture for 36 hours. Peripheral rods and fruiting body spores were separated and examined by fluorescence (left images) or DIC (right images) microscopy.
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Figure 6: nfs genes are upregulated late during starvation induced development and expressed only within sporulating cells. A. The starvation-induced developmental program was induced in submerged culture for strains expressing mCherry fused after the promoter from pilA (PpilA::mCherry; strain PH1221), the 500 bp upstream of nfsA (PnfsA::mCherry; strain PH1220) or the vector backbone (vector; strain PH1222) in the DK1622 background. At the indicated times, mCherry fluorescence was recorded in a plate reader. B. Constructs expressing mCherry fused after the promoter from pilA (PpilA::mCherry; strain PH1221), the 500 bp upstream of nfsA (PnfsA::mCherry; strain PH1220) or the vector backbone (vector; strain PH1222) in the DK1622 background were induced to develop under submerged culture for 36 hours. Peripheral rods and fruiting body spores were separated and examined by fluorescence (left images) or DIC (right images) microscopy.

Mentions: To examine the expression pattern of the nfs mutants during starvation induced development, we next measured the relative mCherry fluorescence of the PnfsA::mCherry, PpilA::mCherry and vector control strains by developing the cells in submerged culture in dishes that could be inserted directly into a plate reader and thus measured fluorescence at various times during the developmental program. The PpilA::mCherry fusion was detected from 0 hours and gradually increased 2.5-fold at 36 hours of development (Figure 6A). In contrast, the PnfsA::mCherry fusion began to be detected between 12-18 hours of development increasing to 6.5 times at 36 hours. There was a gradual accumulation of autofluorescence in the vector control strain (Figure 6A) which may correspond to build up of exopolysaccharides.


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)

nfs genes are upregulated late during starvation induced development and expressed only within sporulating cells. A. The starvation-induced developmental program was induced in submerged culture for strains expressing mCherry fused after the promoter from pilA (PpilA::mCherry; strain PH1221), the 500 bp upstream of nfsA (PnfsA::mCherry; strain PH1220) or the vector backbone (vector; strain PH1222) in the DK1622 background. At the indicated times, mCherry fluorescence was recorded in a plate reader. B. Constructs expressing mCherry fused after the promoter from pilA (PpilA::mCherry; strain PH1221), the 500 bp upstream of nfsA (PnfsA::mCherry; strain PH1220) or the vector backbone (vector; strain PH1222) in the DK1622 background were induced to develop under submerged culture for 36 hours. Peripheral rods and fruiting body spores were separated and examined by fluorescence (left images) or DIC (right images) microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: nfs genes are upregulated late during starvation induced development and expressed only within sporulating cells. A. The starvation-induced developmental program was induced in submerged culture for strains expressing mCherry fused after the promoter from pilA (PpilA::mCherry; strain PH1221), the 500 bp upstream of nfsA (PnfsA::mCherry; strain PH1220) or the vector backbone (vector; strain PH1222) in the DK1622 background. At the indicated times, mCherry fluorescence was recorded in a plate reader. B. Constructs expressing mCherry fused after the promoter from pilA (PpilA::mCherry; strain PH1221), the 500 bp upstream of nfsA (PnfsA::mCherry; strain PH1220) or the vector backbone (vector; strain PH1222) in the DK1622 background were induced to develop under submerged culture for 36 hours. Peripheral rods and fruiting body spores were separated and examined by fluorescence (left images) or DIC (right images) microscopy.
Mentions: To examine the expression pattern of the nfs mutants during starvation induced development, we next measured the relative mCherry fluorescence of the PnfsA::mCherry, PpilA::mCherry and vector control strains by developing the cells in submerged culture in dishes that could be inserted directly into a plate reader and thus measured fluorescence at various times during the developmental program. The PpilA::mCherry fusion was detected from 0 hours and gradually increased 2.5-fold at 36 hours of development (Figure 6A). In contrast, the PnfsA::mCherry fusion began to be detected between 12-18 hours of development increasing to 6.5 times at 36 hours. There was a gradual accumulation of autofluorescence in the vector control strain (Figure 6A) which may correspond to build up of exopolysaccharides.

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