devIIs an Evolutionarily Young Negative Regulator of Myxococcus xanthus Development

Abstract

ABSTRACTDuring starvation-induced development ofMyxococcus xanthus, thousands of rod-shaped cells form mounds in which they differentiate into spores. Thedevlocus 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 indevSordevRof a lab reference strain permit mound formation but impair sporulation. We report that natural isolates ofM. xanthuscapable of normal development are highly polymorphic in the promoter region of thedevoperon. We show that thedevpromoter is predicted to be nonfunctional in most natural isolates and is dispensable for development of a laboratory reference strain. Moreover, deletion of thedevpromoter or the small gene immediately downstream of it, here designateddevI(developmentinhibitor), suppressed the sporulation defect ofdevSordevRmutants in the lab strain. Complementation experiments and the result of introducing a premature stop codon indevIsupport a model in which DevRS proteins negatively autoregulate expression ofdevI, 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 thatdevIis entirely absent from mostM. xanthusnatural 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 thedevoperon and its mechanistic role inM. xanthussporulation.IMPORTANCECertain mutations in thedevCRISPR-Cas (clustered regularly interspaced short palindromic repeat-associated) system ofMyxococcus xanthusimpair 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 functionaldevpromoter, yet these strains sporulate normally. Deletion of thedevpromoter or the small gene downstream of it suppressed the sporulation defect of a lab strain with mutations indevgenes encoding Cas proteins. The results support a model in which the Cas proteins DevRS prevent overexpression of the small genedevI, which codes for an inhibitor of sporulation. Phylogenetic analysis of natural isolates suggests thatdevIand thedevpromoter were only recently acquired in some lineages.