The dev Operon Regulates the Timing of Sporulation during Myxococcus xanthus Development

Abstract

Myxococcus xanthus undergoes multicellular development when starved. Thousands of rod-shaped cells coordinate their movements and aggregate into mounds in which cells differentiate into spores. Mutations in the dev operon impair development. The dev operon encompasses a clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) system. Null mutations in devI, a small gene at the beginning of the dev operon, suppress the developmental defects caused by null mutations in the downstream devR and devS genes but failed to suppress defects caused by a small in-frame deletion in devT. We provide evidence that the original mutant has a second-site mutation. We show that devT null mutants exhibit developmental defects indistinguishable from devR and devS null mutants, and a null mutation in devI suppresses the defects of a devT null mutation. The similarity of DevTRS proteins to components of the CRISPR-associated complex for antiviral defense (Cascade), together with our molecular characterization of dev mutants, support a model in which DevTRS form a Cascade-like subcomplex that negatively autoregulates dev transcript accumulation and prevents DevI overproduction that would strongly inhibit sporulation. Our results also suggest that DevI transiently inhibits sporulation when regulated normally. The mechanism of transient inhibition may involve MrpC, a key transcription factor, whose translation appears to be weakly inhibited by DevI. Finally, our characterization of a devI devS mutant indicates that very little exo transcript is required for sporulation, which is surprising since Exo proteins help form the polysaccharide spore coat.IMPORTANCE CRISPR-Cas systems typically function as adaptive immune systems in bacteria. The dev CRISPR-Cas system of M. xanthus has been proposed to prevent bacteriophage infection during development, but how dev controls sporulation has been elusive. Recent evidence supported a model in which DevR and DevS prevent overproduction of DevI, a predicted 40-residue inhibitor of sporulation. We provide genetic evidence that DevT functions together with DevR and DevS to prevent DevI overproduction. We also show that spores form about 6 h earlier in mutants lacking devI than in the wild type. Only a minority of natural isolates appear to have a functional dev promoter and devI, suggesting that a functional dev CRISPR-Cas system evolved recently in niches where delayed sporulation and/or protection from bacteriophage infection proved advantageous.KEYWORDS CRISPR-Cas, Myxococcus xanthus, bacterial development, dev operon, gene regulation, signaling, sporulation M yxococcus xanthus is a Gram-negative soil bacterium that exhibits social behaviors (1). In nature, cells of M. xanthus move in groups, feeding on prey bacteria and organic detritus. When starved, thousands of cells aggregate to form mounds that mature into fruiting bodies as rod-shaped cells differentiate into dormant ovoid spores.

Systematic analysis of theMyxococcus xanthusdevelopmental gene regulatory network supports posttranslational regulation of FruA by C-signaling

“…The dev mRNA level ranged 249 from 20-fold higher in the devS mutant than in WT at 18 h PS, to 10-fold higher at 30 h PS ( Fig. 4A), 250 consistent with negative autoregulation by DevS (and DevT and DevR) reported previously 251 (Rajagopalan & Kroos, 2017, Rajagopalan et al, 2015. Unexpectedly, the dev mRNA level in the devI 252 mutant was about threefold lower than in WT at 30 h PS ( Fig.…”

“…S1B). The devI mutant was similar to WT, except spores were first observed 6 h earlier at 21 h 160 PS, as reported recently (Rajagopalan & Kroos, 2017). The csgA, fruA, and devS mutants failed to make 161 a detectable number of spores (at a detection limit of 0.04% of the T0 number) and appeared to be 162 slightly delayed relative to WT and the devI mutant in terms of the declining number of sonication- 163 sensitive cells ( Fig.…”

“…4A), suggesting that negative 569 autoregulation mediated by DevS has a stronger effect leading up to the commitment period than 570 during commitment. Strong negative autoregulation may promote commitment to sporulation by 571 lowering the level of DevI, which would raise the MrpC level by relieving inhibition of translation of 572 mrpC transcripts (Rajagopalan & Kroos, 2017). Our data suggest that negative autoregulation by 573 DevTRS weakens during the commitment period, perhaps accounting for the observed small rise in the 574 dev transcript level (Fig.…”

“…198 S3B), consistent with the finding that the 16S rRNA level does not change significantly, since the 199 majority of total RNA is rRNA. 200 To measure protein levels, a portion of each well-mixed developing population was immediately 201 added to sample buffer, boiled, and frozen for subsequent semi-quantitative immunoblot analysis 202 (Rajagopalan & Kroos, 2017). The rest of the population was used for cell counting and RNA analysis as 206 By systematically quantifying protein and mRNA levels during the period leading up to and including 207 the time that cells commit to spore formation, we investigated whether the GRN shown in Figure 1 208 could account for observed changes over time in WT and in mutants.…”

A negative autoregulation network motif is required for synchronizedMyxococcus xanthusdevelopment

“…Early during the developmental program, gradual accumulation of MrpC is achieved because the Esp signaling system induces turnover of MrpC (22, 23). Several additional post-transcriptional events modulate MrpC accumulation (and therefore progression through development) in response to changing environmental conditions (18, 19, 24). A long-standing model suggested MrpC acted as positive autoregulator, leading to the attractive speculation that bifurcation of MrpC production (a known feature of positive autoregulation motifs (25)) could drive cell fate segregation into either aggregation centers or peripheral rods [summarized in (15)].…”