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. 2015 Apr;197(7):1249-62.
doi: 10.1128/JB.02542-14. Epub 2015 Feb 2.

devI is an evolutionarily young negative regulator of Myxococcus xanthus development

Ramya Rajagopalan  1 , Sébastien Wielgoss  2 , Gerardo Lippert  2 , Gregory J Velicer  2 , Lee Kroos  3
Affiliations

devI is an evolutionarily young negative regulator of Myxococcus xanthus development

Ramya Rajagopalan et al. J Bacteriol. 2015 Apr.

Abstract

During starvation-induced development of Myxococcus xanthus, thousands of rod-shaped cells form mounds in which they differentiate into spores. The dev locus includes eight genes followed by clustered regularly interspaced short palindromic repeats (CRISPRs), comprising a CRISPR-Cas system (Cas stands for CRISPR associated) typically involved in RNA interference. Mutations in devS or devR of a lab reference strain permit mound formation but impair sporulation. We report that natural isolates of M. xanthus capable of normal development are highly polymorphic in the promoter region of the dev operon. We show that the dev promoter is predicted to be nonfunctional in most natural isolates and is dispensable for development of a laboratory reference strain. Moreover, deletion of the dev promoter or the small gene immediately downstream of it, here designated devI (development inhibitor), suppressed the sporulation defect of devS or devR mutants in the lab strain. Complementation experiments and the result of introducing a premature stop codon in devI support a model in which DevRS proteins negatively autoregulate expression of devI, whose 40-residue protein product DevI inhibits sporulation if overexpressed. DevI appears to act in a cell-autonomous manner since experiments with conditioned medium and with cell mixtures gave no indication of extracellular effects. Strikingly, we report that devI is entirely absent from most M. xanthus natural isolates and was only recently integrated into the developmental programs of some lineages. These results provide important new insights into both the evolutionary history of the dev operon and its mechanistic role in M. xanthus sporulation.

Importance: Certain mutations in the dev CRISPR-Cas (clustered regularly interspaced short palindromic repeat-associated) system of Myxococcus xanthus impair sporulation. The link between development and a CRISPR-Cas system has been a mystery. Surprisingly, DNA sequencing of natural isolates revealed that many appear to lack a functional dev promoter, yet these strains sporulate normally. Deletion of the dev promoter or the small gene downstream of it suppressed the sporulation defect of a lab strain with mutations in dev genes encoding Cas proteins. The results support a model in which the Cas proteins DevRS prevent overexpression of the small gene devI, which codes for an inhibitor of sporulation. Phylogenetic analysis of natural isolates suggests that devI and the dev promoter were only recently acquired in some lineages.

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Figures

FIG 1
FIG 1
Map of the M. xanthus laboratory strain DK1622 dev operon and structural evolution of the dev promoter region among natural lineages. (A) Map of the DK1622 dev operon and expanded view of the dev promoter region. The top part depicts the eight genes of the dev operon, which also includes at least two repeats of the downstream CRISPR. The expanded view shows the region of some natural isolates that was subjected to Sanger sequencing (sequences are shown in Fig. S1 in the supplemental material). This region includes the dev promoter and the small gene devI. Numbers are relative to the transcriptional start site (+1), and a bent arrow indicates the direction of transcription. (B) Structure and evolution of the dev promoter region. The leftward part shows phylogenetic relationships among natural isolates inferred from ∼4.5 Mbp of orthologous sequence. M. fulvus HW-1 (52) served as the outgroup. Bootstrap values supporting branch inferences are shown near each branch. The center part shows the structure of the dev promoter region. Black boxes highlight orthologous regions shared among natural isolates and the reference strain DK1622. Thin lines depict DK1622 sequence regions that are absent from the respective genomes, either because there is no sequence at all in the corresponding region or because the sequence that is present is nonorthologous to that of DK1622. The rightward part shows a phylogenetic tree of a conserved partial segment of MXAN_7267 highlighting recent evolutionary history of the orthologous coding region upstream of the dev promoter. Bootstrap values larger than 60% are shown at the respective nodes. Filled circles indicate strains identified by the horizontally corresponding strain in the leftward phylogeny, whereas open circles identify strains found at a different horizontal level in the leftward phylogeny. Note that the topological structures of MXAN_7267- and whole-genome-based trees are divergent. Branches of the MXAN_7267 tree were rotated around nodes to maximize horizontal alignment to the genome-based phylogeny, but these rotations did not alter tree structure. The asterisk on the branch leading to Chihaya 20 represents the most likely branch position of DK897 in the MXAN_7267 tree, based on the available sequence information for DK897 (see Fig. S1 in the supplemental material), which covered significantly less of the conserved partial segment of MXAN_7267 than it did for the other strains and therefore was not used in the tree construction.
FIG 2
FIG 2
Development of dev mutants under submerged culture conditions. (A) Fruiting body formation by the laboratory strain DK1622 and the indicated dev mutants. Dark fruiting bodies are observed at 96 h into development of DK1622 (an arrow points to one) but not the dev mutants. Bar, 100 μm. Similar results were observed in at least two biological replicates. (B) Cellular shape change. Developing cultures were subjected to gentle dispersion and examined microscopically at the indicated times. Photos show densely packed cell aggregates presumed to be nascent fruiting bodies. Arrows point to round or ovoid cells. Bar, 5 μm. Similar results were observed in at least two biological replicates. (C) Sporulation. Samples were harvested at 72 h into development for measurement of mature spores. Values (log10) are the averages from at least three biological replicates, and error bars represent 1 standard deviation from the mean.
FIG 3
FIG 3
Development of ΔPdev and ΔdevI mutants. Fruiting body formation by the laboratory strain DK1622 and the indicated mutants under submerged culture conditions. Arrows point to dark fruiting bodies observed at 72 h poststarvation for DK1622 and several of the mutants. Bar, 100 μm. Similar results were observed in at least two biological replicates.
FIG 4
FIG 4
Levels of dev transcripts. (A) Comparison of the laboratory strain DK1622 with the ΔPdev mutant. At 24 h poststarvation under submerged culture conditions, cultures were harvested, RNA was isolated, and the RNA was subjected to qRT-PCR analysis using primers Pdev-T-F and Pdev-T-R, designed to amplify the region from +4811 to +4940 relative to the transcriptional start site of the dev operon. (B) Comparison of DK1622 with the ΔdevS and ΔdevR mutants. Culture conditions were as described for panel A. RNA was subjected to qRT-PCR analysis using primers PdevF-2 and PdevR-3, designed to amplify the region from +237 to +440 relative to the dev transcriptional start site. For both panels, values are the averages of three technical replicates for at least three biological replicates and are reported relative to DK1622. Error bars indicate 1 standard deviation from the mean.
FIG 5
FIG 5
Complementation of the ΔdevI ΔdevS double mutant. (A) Map of the dev promoter region and fragments used for complementation. Boxes indicate genes. Numbers are relative to the transcriptional start site (+1), and a bent arrow indicates the direction of transcription. Lines below the map indicate fragments used for complementation. (B) Fruiting body formation by the laboratory strain DK1622 and the indicated mutants under submerged culture conditions. Endpoints of complementing segments relative to the dev transcriptional start site are indicated in parentheses. Arrows point to dark fruiting bodies observed at 72 h poststarvation for DK1622 and several of the mutants. Bar, 100 μm. Similar results were observed in at least two biological replicates. (C) Sporulation. Samples were harvested at 72 h into development for measurement of mature spores. Values (log10) are the averages from at least three biological replicates, and error bars represent 1 standard deviation from the mean. (D) Levels of dev transcripts. At 24 h poststarvation under submerged culture conditions, cultures were harvested, RNA was isolated, and the RNA was subjected to qRT-PCR analysis using primers 7266-F and 7266-R, designed to amplify the region from +36 to +159. Values are the averages from three technical replicates for at least three biological replicates and are reported relative to DK1622. Error bars indicate 1 standard deviation from the mean.
FIG 6
FIG 6
Effects of a premature stop codon in devI. (A) Fruiting body formation by the laboratory strain DK1622 and the indicated mutants under submerged culture conditions. devI-STOP indicates the single-base-pair insertion that creates an in-frame stop codon. Arrows point to dark fruiting bodies observed at 72 h poststarvation. Bar, 100 μm. Similar results were observed in at least two biological replicates. (B) Sporulation. Samples were harvested at 96 h into development for measurement of mature spores. Values (log10) are the averages from at least three biological replicates, and error bars represent 1 standard deviation from the mean.
FIG 7
FIG 7
Effect of conditioned starvation buffer from the ΔdevS mutant on development of the laboratory strain DK1622 and effect of codevelopment of the ΔdevS mutant with DK1622. (A) Overlying conditioned starvation buffer from the ΔdevS mutant at 18, 24, or 30 h under submerged culture conditions was used to replace the overlying buffer of comparably treated DK1622 at 18 or 24 h poststarvation (e.g., 24-18 means the conditioned overlay from ΔdevS at 24 h was used to replace the overlay of DK1622 at 18 h). Samples collected at 30 and 36 h poststarvation (left and right bars, respectively, for each replacement regimen) were sonicated, and spores were quantified microscopically using a Neubauer counting chamber. Values (log10) are the averages from at least three biological replicates. Error bars represent 1 standard deviation from the mean. (B) Effect of codevelopment on fruiting body formation. A derivative of strain DK1622 with a kanamycin resistance marker (DK1622 Kmr), the ΔdevS mutant, and mixtures of the two strains at the indicated ratios were subjected to starvation under submerged culture conditions. Arrows point to dark fruiting bodies observed at 96 h poststarvation. Bar, 100 μm. Similar results were observed in at least two biological replicates. (C) Effect of codevelopment on sporulation. Samples were harvested at 96 h for the measurement of mature spores. Values (log10) are the averages from at least three biological replicates, and error bars represent 1 standard deviation from the mean.
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