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Transcriptome Dynamics of the Myxococcus xanthus Multicellular Developmental Program

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Transcriptome Dynamics of the Myxococcus xanthus Multicellular Developmental Program

José Muñoz-Dorado et al. Elife.

Abstract

The bacterium Myxococcus xanthus exhibits a complex multicellular life cycle. In the presence of nutrients, cells prey cooperatively. Upon starvation, they enter a developmental cycle wherein cells aggregate to produce macroscopic fruiting bodies filled with resistant myxospores. We used RNA-Seq technology to examine the transcriptome of the 96 hr developmental program. These data revealed that 1415 genes were sequentially expressed in 10 discrete modules, with expression peaking during aggregation, in the transition from aggregation to sporulation, or during sporulation. Analysis of genes expressed at each specific time point provided insights as to how starving cells obtain energy and precursors necessary for assembly of fruiting bodies and into developmental production of secondary metabolites. This study offers the first global view of developmental transcriptional profiles and provides important tools and resources for future studies.

Keywords: Myxococcus xanthus; development; infectious disease; microbiology; transcriptome.

Conflict of interest statement

JM, AM, FM, FC, AM, JS, PH, JP No competing interests declared

Figures

Figure 1.
Figure 1.. Schematic of the M. xanthus developmental program.
The time line indicates aggregation and sporulation phases. M. xanthus cells (yellow rods) aggregate into mounds (arrows indicate gliding to aggregation centers) and then differentiate into resistant spores (gray circles) to produce mature fruiting bodies. Peripheral rods (gray rods) remain outside of the fruiting bodies as a distinct differentiated state. Cells undergoing lysis are depicted with dashed lines.
Figure 2.
Figure 2.. Validation of the RNA-Seq transcription patterns for genes spiA (MXAN_RS20760) (A) and fmgE (MXAN_RS16790) (B).
β-galactosidase specific activity (SA) of the strains harboring lacZ fusions to the respective genes (blue lines) compared to RNA-Seq RPKM values (red lines) at each developmental time point (h). Error bars indicate standard deviations for β-galactosidase specific activity determination.
Figure 3.
Figure 3.. The relative expression profiles of M. xanthus genes observed during the developmental program compared to those previously observed during chemical-induction of sporulation.
(A) Relative expression profiles of significantly regulated genes at the indicated hours after induction of starvation. Genes were clustered into 10 developmental groups based on the time of peak expression and then organized according to the temporal progression of development. Developmental group number and the phase of the developmental program (with photographs of aggregates under the dissecting microscope and cells under the scanning electron microscope) are indicated to the left of the heat map. In the photographs, red, blue and yellow bars represent 2 mm, 100 µm, and 5 µm, respectively. (B) Relative expression levels of the genes in panel A during the indicated hours after chemical-induction of sporulation (Müller et al., 2010). The position of individual genes in panel B is matched to panel A. Relative expression levels for panels A and B are indicated by color code according to the legend. DGs significantly represented in up-, down-, or not-regulated sporulation gene sets are indicated by the probability values in yellow, blue or gray, respectively (C) Comparison of up-, down-, or not-regulated starvation-induced development and chemical-induced sporulation gene tallies. Tally of glycerol-induced sporulation genes up- (top), down- (middle) or not-regulated (bottom) that are significantly enriched in the indicated DGs.
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. Relative expression profiles of significantly regulated genes at the indicated hours after induction of starvation.
Genes were clustered into a different number of developmental groups to decide which number of groups is the most appropriate.
Figure 3—figure supplement 2.
Figure 3—figure supplement 2.. Tally of reliable genes.
Number (#) genes with >50 reads for each time point and replicate correlations > 0.7). Bars are colored coded based on the indicated stages of development assigned in Figure 3. Bar height is proportional to number of genes. DG, developmental group; const, constitutive expression.
Figure 3—figure supplement 3.
Figure 3—figure supplement 3.. Number of genes composing 50% of the transcriptome throughout the developmental program.
Figure 3—figure supplement 4.
Figure 3—figure supplement 4.. A: Relationship between M. xanthus developmental phases, developmental groups, and a known functional interaction network.
(A) Developmental clusters with peak expressions corresponding to the indicated developmental phases. Size of the bars correspond to the number of genes in each category (see Figure 3—source data 1). Yellow bars, peak expression during growth. Blue bars, peak expression during aggregation; green bars, peak expression during transition from aggregation to sporulation; gray bars, peak expression during sporulation phases. (B) Limited functional interaction network of certain well defined genes and their known regulatory networks. Proteins/genes are color coded to the developmental phase from A and aligned with the specific DG in which they were identified. Gray proteins did not meet quality control criteria. Blue text represents small molecule or protein signals. Red proteins were identified in DGs with peak expression profiles that did not match their predicted function. The maroon inset is expanded below the figure. Arrows indicate positive regulation and blunt-ended lines indicate negative regulation.
Figure 4.
Figure 4.. Developmental expression levels of M. xanthus motility proteins.
Schematic representation of the focal adhesion motor complexes necessary for A motility (top), the type IV pili motor complexes necessary for S motility (bottom), and the proteins involved in controlling polarity of both engines (polarity control module; center). The developmental expression levels (RPKM) of significantly regulated motility genes at the indicated times (h) of development are depicted. Gene expression profiles are colored to match the proteins shown in the schematic. Proteins depicted in gray represent genes that were not included in the developmental groups.
Figure 5.
Figure 5.. Relative developmental expression profiles of genes involved in energy generation.
(A) Genes encoding protein homologs for the pyruvate dehydrogenase complex, TCA cycle, and oxidative phosphorylation proteins. (B) Genes necessary for glycolysis/gluconeogenesis. Developmental time points in hours are indicated above each panel. Relative expression levels for panels A and B are indicated by color code according to the legend. For simplicity, the MXAN_RS designation was omitted from the locus tag of each gene.
Figure 6.
Figure 6.. Genes involved in synthesis and degradation of lipids.
(A) Simple representation of the M. xanthus branched fatty acid metabolic pathways depicting leucine degradation and alternative mevalonate-dependent routes. (B) Relative developmental expression profiles of the genes involved in straight-chain and branched-chain fatty acid biosynthesis as designated to the right. (B1) Straight-chain fatty acid primer synthesis; (B2) Branched-chain fatty acid primer synthesis of isovaleryl-CoA via leucine degradation (bkd genes); (B3) Branched-chain fatty acid primer synthesis of isovaleryl-CoA via the alternative pathway (mevalonate); B4: Fatty acid elongation. (C) Lipid degradation via β oxidation (C1) and other pathways (C2). Relative expression levels for panels B and C are indicated by color code according to the legend and developmental time points in hours are indicated above each panel. The MXAN_RS designation was omitted from the locus tag of each gene.
Figure 7.
Figure 7.. Developmental expression profiles of genes involved in production of polysaccharides and proteins.
Relative expression profiles of genes predicted to be necessary for polysaccharide hydrolysis (A), polysaccharide synthesis (B), and encoding proteases and peptidases (C). Developmental time points in hours are indicated above each panel and relative expression levels are indicated by color code according to the legend at the bottom. The MXAN_RS designation was omitted from the locus tag of each gene.
Figure 8.
Figure 8.. Developmental expression levels of genes involved in myxovirescine (antibiotic TA), myxoprincomide and other secondary metabolite biosynthesis.
(A) Schematic of the myxovirescine gene cluster. Names of genes depicted here are ta followed by the capital letter written with each arrow. (B) and (C) Developmental expression levels (RPKM) of significantly regulated myxovirescine genes plotted against the indicated developmental time points in hours. Gene expression profiles are colored to match the genes depicted in panel A. Genes depicted in gray were not included in the developmental groups. (D) Expression profiles of genes involved in myxoprincomide biosynthesis. (E) Profiles of others NRPS, PKS and PKS/NRPS not included in panels C and D. The MXAN_RS designation was omitted from the locus tag of each gene.
Figure 9.
Figure 9.. Developmental expression profiles of genes involved in protein production.
(A) Relative expression levels of genes involved in translation or ribosome assembly. (B) Relative expression levels of genes encoding ribosomal proteins. Relative expression levels for panels A and B are indicated by color code according to the legend at the bottom, and developmental time points in hours are indicated above each panel. The MXAN_RS designation was omitted from the locus tag of each gene. (C) Developmental expression levels (RPKM) of the paralogous genes encoding protein S4 plotted against developmental time points in hours.
Figure 10.
Figure 10.. Developmental expression profiles of genes involved in transcriptional regulation and signal transduction.
Relative expression levels of genes encoding one-component regulators (A), two-component signal transduction proteins (B), sigma factors (C), and serine/threonine protein kinases (F) are depicted. Relative expression levels for panels A, B, C, and F are indicated by color code according to the legend at the bottom, and developmental time points in hours are indicated above each panel. The MXAN_RS designation was omitted from the locus tag of each gene. Expression levels (RPKM) of genes encoding the major sigma factors (D) and the subunits of the RNA polymerase (E) plotted against developmental time points in hours.
Figure 11.
Figure 11.. New signaling proteins that are developmentally regulated in M. xanthus identified in this study.
Code used to distinguish among types of regulators is indicated in the upper part, where OCS indicates one-component systems; HK, histidine kinase: hyHK, hybrid histidine kinase; CheY, CheY-likeresponse regulator; RR, response regulator; ECF, ECF sigma factor; STPK, active Ser/Thr protein kinase; PseudoK, pseudokinase. Numbers inside each symbol indicate the number of each type of regulator that have not been previously identified as being developmentally regulated. Information about proteins depicted here is shown in Figure 11—source data 1.

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