Spontaneous Reversions of an Evolutionary Trait Loss Reveal Regulators of a Small RNA That Controls Multicellular Development in Myxobacteria

Abstract

Lost traits can reevolve, but the probability of trait reversion depends partly on a trait's genetic complexity. Myxobacterial fruiting body development is a complex trait controlled by the small RNA (sRNA) Pxr, which blocks development under conditions of nutrient abundance. In developmentally proficient strains of Myxococcus xanthus, starvation relaxes the inhibition by Pxr, thereby allowing development to proceed. In contrast, the lab-evolved strain OC does not develop because it fails to relay an early starvation signal that alleviates inhibition by Pxr. A descendant of OC, strain PX, previously reevolved developmental proficiency via a mutation in pxr that inactivates its function. A single-colony screen was used to test whether reversion of OC to developmental proficiency occurs only by mutation of pxr or might also occur through alternative regulatory loci. Five spontaneous mutants of OC that exhibited restored development were isolated, and all five showed defects in Pxr synthesis, structure, or processing, including one that incurred an eight-nucleotide deletion in pxr. Two mutations occurred in the 54 response regulator (RR) gene MXAN_1078 (named pxrR here), immediately upstream of pxr. PxrR was found to positively regulate pxr transcription, presumably via the 54 promoter of pxr. Two other mutations were identified in a histidine kinase (HK) gene (MXAN_1077; named pxrK here) immediately upstream of pxrR. Evolutionarily, the rate of trait restoration documented in this study suggests that reversion of social defects in natural microbial populations may be common. Molecularly, these results suggest a mechanism by which the regulatory functions of an HK-RR two-component signaling system and an sRNA are integrated to control initiation of myxobacterial development. IMPORTANCEMany myxobacteria initiate a process of multicellular fruiting body development upon starvation, but key features of the regulatory network controlling the transition from growth to development remain obscure. Previous work with Myxococcus xanthus identified the first small RNA (sRNA) regulator (Pxr) known to serve as a gatekeeper in this life history transition, as it blocks development when nutrients are abundant. In the present study, a screen for spontaneous mutants of M. xanthus was developed that revealed a two-component system operon (encoding a histidine kinase and a 54 response regulator) associated with the production and processing of Pxr sRNA. This discovery broadens our knowledge of early developmental gene regulation and also represents an evolutionary integration of two-component signaling and sRNA gene regulation to control a bacterial social trait. Functional traits are often lost from evolving populations after selection favoring those traits has been relaxed (1-4). Although Dollo famously asserted that evolutionary losses are irreversible (5), lost traits might be restored evolutionarily, even if infrequently (6). Among animals, some species of dust mites appear to have reevolved host independence from ances...

The path to re-evolve cooperation is constrained in Pseudomonas aeruginosa

“…It is well known that mutations causing a loss of function are disproportionately more likely to occur than mutations resulting in a gain of function [28]. On the other hand, there are a few examples of evolutionary trait restoration through mutation in microbes [29][30][31]. In the context of our experiment, re-evolution of pyoverdine production could have happened by a reversion to the ancestral PAO1 genotype (i.e.…”

sRNA‐pathway genes regulating myxobacterial development exhibit clade‐specific evolution

“…Transposon insertions from candidate mutants were backcrossed into the parental strain GVB207.3 to confirm that positive developmental phenotypes were linked only with a single transposon insertion rather than spontaneous mutations (Yu et al, 2016) or multiple transposon insertions. To backcross insertions into GVB207.3, genomic DNA of each transposon mutant was isolated from vegetative cultures following a previous protocol (Wu & Kaiser, 1995).…”

“…To reconstruct the phylogeny of these species and strains, DNA sequences of their 5S, 16S and FIGURE 1 Mapping strategy to identify locations of insertion loci in transposon mutants. For comparison, we also examined the protein sequences of three conserved loci (pyrG, rpoB and pgm) used in the phylogenetic reconstruction in the myxobacteria (Chen, Griesenauer, Yu, & Velicer, 2014) and the loci in a twocomponent regulatory system (pxrK and pxrR) involved in the synthesis and processing of Pxr (Yu et al, 2016). "Tn" refers to the Himar1-derived transposon.…”

“…Such processingmediated Pxr function is analogous to the generation of mature eukaryotic miRNAs and siRNAs (Shukla, Singh, & Barik, 2011). The pxr gene appears to be transcribed from an upstream σ 54 promoter and is located in the intergenic region downstream of a two-component system operon (a histidine kinase gene pxrK (Mxan_1077) and the σ 54 -dependent response regulator pxrR (aka nla19 or Mxan_1078)) (Yu, Kleiner, & Velicer, 2016;Yu et al, 2010). The pxr gene appears to be transcribed from an upstream σ 54 promoter and is located in the intergenic region downstream of a two-component system operon (a histidine kinase gene pxrK (Mxan_1077) and the σ 54 -dependent response regulator pxrR (aka nla19 or Mxan_1078)) (Yu, Kleiner, & Velicer, 2016;Yu et al, 2010).…”

“…Upon starvation of M. xanthus wild-type cells, Pxr-S levels rapidly and greatly decrease, hence alleviating blockage of development. This pxrK/pxrR regulatory module is required for the synthesis and processing of Pxr (Yu et al, 2016). This pxrK/pxrR regulatory module is required for the synthesis and processing of Pxr (Yu et al, 2016).…”