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

Abstract

Small non-coding RNAs (sRNAs) control bacterial gene expression involved in a wide range of important cellular processes. In the highly social bacterium Myxococcus xanthus, the sRNA Pxr prevents multicellular fruiting-body development when nutrients are abundant. Pxr was discovered from the evolution of a developmentally defective strain (OC) into a developmentally proficient strain (PX). In OC, Pxr is constitutively expressed and blocks development even during starvation. In PX, one mutation deactivates Pxr allowing development to proceed. We screened for transposon mutants that suppress the OC defect and thus potentially reveal new Pxr-pathway components. Insertions significantly restoring development were found in four genes-rnd, rnhA, stkA and Mxan_5793-not previously associated with an sRNA activity. Phylogenetic analysis suggests that the Pxr pathway was constructed within the Cystobacterineae suborder both by co-option of genes predating the Myxococcales order and incorporation of a novel gene (Mxan_5793). Further, the sequence similarity of rnd, rnhA and stkA homologs relative to M. xanthus alleles was found to decrease greatly among species beyond the Cystobacterineae suborder compared to the housekeeping genes examined. Finally, ecological context differentially affected the developmental phenotypes of distinct mutants, with implications for the evolution of development in variable environments.

Mutation of rpoB Shifts the Nutrient Threshold Triggering Myxococcus Multicellular Development

“…Therefore, undergoing development while nutrients are still abundant would seem undesirable. Previous work from our group has identified several genes that regulate the transition to development, all of which appear to belong to the same regulatory pathway controlled by the small RNA Pxr ( Yu et al, 2010 , 2016 ; Chen et al, 2019 ). In this study, we identify a mutation in the universal bacterial transcription machinery gene rpoB that impacts the M. xanthus developmental response to nutrients, a connection not revealed by mutations previously known to confer RM development.…”

Ribonuclease D Processes a Small RNA Regulator of Multicellular Development in Myxobacteria

“…Our previous study found that the suppressor strain OC::TnE1 was able to form sporebearing fruiting bodies despite its parental strain's developmental defect [26]. Figure 2 shows the defect of the parental strain OC compared to the restored ability of OC::TnE1 to form fruiting bodies.…”

“…OC is unable to eliminate Pxr-S in response to starvation [22] and is therefore deficient in starvation-induced fruiting-body formation in pure culture. OC::TnE1 was found by screening a transposon-mutant library for restored developmental proficiency and has a transposon inserted in the gene MXAN_5981 (rnd), a Ribonuclease D homolog [26]. GJV1::TnE1 and OC::TnE1-r were constructed by transforming a fresh growing strain of GJV1 and GVB207.3, respectively, with ~500 ng of OC::TnE1 genomic DNA and plated on CTT-kanamycin to select for clones resulting from a double crossover at the homologous regions flanking the transposon interstation site.…”

“…The absence of functional Pxr appears to be essential for suppressing the OC phenotype. To further expand our search for genetic elements essential for the Pxr regulatory pathway, we conducted a screen of transposon-insertion mutants of OC and identified mutants that can undergo development [26]. We hypothesized that Tn insertions in genes important for the Pxr pathway will alleviate its negative regulation and allow cells to initiate fruiting-body formation.…”

“…Indeed, several Tn mutants exhibited developmentally proficient phenotypes, including one designated TnE1. The TnE1 insertion locus has been mapped to rnd (MXAN_5981), which is predicted to encode Ribonuclease D (RNase D) [26]. A major function of the prokaryotic RNase D is to process the 3 end of tRNA precursors [27,28].…”