devIIs an Evolutionarily Young Negative Regulator of Myxococcus xanthus Development

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 cellautonomous 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. IMPORTANCECertain 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. Myxococcus xanthus is a Gram-negative bacterium of the deltaproteobacteria group. Typically found in the soil, M. xanthus exhibits social behaviors (1). The rod-shaped cells move in groups on solid surfaces, feeding on prey bacteria and organic matter. When starved, cells modify their movements and build mounds containing thousands of individuals that differentiate into ovoid spores. The resulting mound of dormant and insultresistant spores is called a fruiting body. Spores within the fruiting body germinate when con...

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

“…Another open question from the previous study is the role of DevT during development (10). The developmental defect of a devT mutant was more severe than those of devR and devS mutants, and the devT defect was not overcome by deletion of the dev promoter or devI.…”

“…Since the dev operon is a direct target of transcriptional activation by MrpC and FruA (9), a simple model proposes that dev expression governs commitment to sporulation (5). However, recent work showed that deletion of the dev promoter or the first gene of the operon, devI, suppresses the sporulation defects of devR and devS mutants (10). Since devR and devS mutants accumulate more dev transcripts than the wild type, it was proposed that overexpression of devI in the mutants inhibits sporulation.…”

“…(ΔdevT408 -502 mutant) failed to form mounds in submerged culture, and its developmental defect was not suppressed by a deletion of the dev promoter (ΔP dev ) or devI (10). Since ΔdevR and ΔdevS mutants form mounds but very few spores, and this defect was suppressed by ΔP dev and ΔdevI deletions, we questioned whether the ΔdevT408 -502 mutant had acquired a second-site mutation that affects development.…”

“…The devT mutant studied previously has a small in-frame deletion (6,10), so it is possible that a DevT variant with altered activity is produced. Alternatively, it is possible that the devT mutant acquired a second-site mutation that affects development (10). Here, we clarify the role of DevT in development.…”

“…An alternative role of CRISPR-Cas systems in bacteria is in endogenous gene regulation (12). Negative autoregulation by devRS provided one of the first examples of this role (7,8,10) (Fig. 1).…”