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

By targeting mRNA transcripts, non-coding small RNAs (sRNAs) regulate the expression of genes governing a wide range of bacterial functions. In the social myxobacterium Myxococcus xanthus, the sRNA Pxr serves as a gatekeeper of the regulatory pathway controlling the life-cycle transition from vegetative growth to multicellular fruiting body development. When nutrients are abundant, Pxr prevents the initiation of the developmental program, but Pxr-mediated inhibition is alleviated when cells starve. To identify genes essential for Pxr function, a developmentally defective strain in which Pxr-mediated blockage of development is constitutively active (strain "OC") was transposon-mutagenized to identify suppressor mutations that inactivate or bypass Pxr inhibition and thereby restore development. One of the four loci in which a transposon insertion restored development is rnd, encoding the Ribonuclease D protein (RNase D). RNase D is an exonuclease important for tRNA maturation. Here, we show that disruption of rnd abolishes the accumulation of Pxr-S, the product of Pxr processing from a longer precursor form (Pxr-L) and the active inhibitor of development. Additionally, the decrease in Pxr-S caused by rnd disruption was associated with increased accumulation primarily of a longer novel Pxr-specific transcript (Pxr-XL) rather than of Pxr-L. The introduction of a plasmid expressing rnd reverted cells back to OC-like phenotypes in development and Pxr accumulation, indicating that a lack of RNase D alone suppresses the developmental defect of OC. Moreover, an in vitro Pxr-processing assay demonstrated that RNase D processes Pxr-XL into Pxr-L; this implies that overall, Pxr sRNA maturation requires a sequential two-step processing. Collectively, our results indicate that a housekeeping ribonuclease plays a central role in a model form of microbial aggregative development. To our knowledge, this is the first evidence implicating RNase D in sRNA processing.

Allopatric divergence of cooperators confers cheating resistance and limits effects of a defector mutation.

BACKGROUND: Social defectors may meet diverse cooperators. Genotype-by-genotype interactions may constrain the ranges of cooperators upon which particular defectors can cheat, limiting cheater spread. Upon starvation, the soil bacterium Myxococcus xanthus cooperatively develops into spore-bearing fruiting bodies, using a complex regulatory network and several intercellular signals. Some strains (cheaters) are unable to sporulate effectively in pure culture due to mutations that reduce signal production but can exploit and outcompete cooperators within mixed groups. RESULTS: In this study, interactions between a cheater disrupted at the signaling gene csgA and allopatrically diversified cooperators reveal a very small cheating range. Expectedly, the cheater failed to cheat on all natural-isolate cooperators owing to non-cheater-specific antagonisms. Surprisingly, some lab-evolved cooperators had already exited the csgA mutant's cheating range after accumulating fewer than 20 mutations and without experiencing cheating during evolution. Cooperators might also diversify in the potential for a mutation to reduce expression of a cooperative trait or generate a cheating phenotype. A new csgA mutation constructed in several highly diverged cooperators generated diverse sporulation phenotypes, ranging from a complete defect to no defect, indicating that genetic backgrounds can limit the set of genomes in which a mutation creates a defector. CONCLUSIONS: Our results demonstrate that natural populations may feature geographic mosaics of cooperators that have diversified in their susceptibility to particular cheaters, limiting defectors' cheating ranges and preventing them from spreading. This diversification may also lead to variation in the phenotypes generated by any given cooperation-gene mutation, further decreasing the chance of a cheater emerging which threatens the persistence of cooperation in the system.

Cooperation and Cheating among Germinating Spores.

Many microbes produce stress-resistant spores to survive unfavorable conditions [1-4] and enhance dispersal [1, 5]. Cooperative behavior is integral to the process of spore formation in some species [3, 6], but the degree to which germination of spore populations involves social interactions remains little explored. Myxococcus xanthus is a predatory soil bacterium that upon starvation forms spore-filled multicellular fruiting bodies that often harbor substantial diversity of endemic origin [7, 8]. Here we demonstrate that germination of M. xanthus spores formed during fruiting-body development is a social process involving at least two functionally distinct social molecules. Using pairs of natural isolates each derived from a single fruiting body that emerged on soil, we first show that spore germination exhibits positive density dependence due to a secreted "public-good" germination factor. Further, we find that a germination defect of one strain under saline stress in pure culture is complemented by addition of another strain that germinates well in saline environments and mediates cheating by the defective strain. Glycine betaine, an osmo-protectant utilized in all domains of life, is found to mediate saline-specific density dependence and cheating. Density dependence in non-saline conditions is mediated by a distinct factor, revealing socially complex spore germination involving multiple social molecules.

Kin discrimination and outer membrane exchange in Myxococcus xanthus: Experimental analysis of a natural population.

In some species of myxobacteria, adjacent cells sufficiently similar at the adhesin protein TraA can exchange components of their outer membranes. The primary benefits of such outer membrane exchange (OME) in natural populations are unclear, but in some OME interactions, transferred OM content can include SitA toxins that kill OME participants lacking an appropriate immunity gene. Such OME-dependent toxin transfer across Myxococcus xanthus strains that differ only in their sitBAI toxin/antitoxin cassette can mediate inter-strain killing and generate colony-merger incompatibilities (CMIs)-inter-colony border phenotypes between distinct genotypes that differ from respective self-self colony interfaces. Here we ask whether OME-dependent toxin transfer is a common cause of prevalent CMIs and antagonisms between M. xanthus natural isolates identical at TraA. We disrupted traA in eleven isolates from a cm-scale soil population and assayed whether traA disruption eliminated or reduced CMIs between swarming colonies or antagonisms between strains in mixed cultures. Among 33 isolate pairs identical at traA that form clear CMIs, in no case did functional disruption of traA in one partner detectably alter CMI phenotypes. Further, traA disruption did not alleviate strong antagonisms observed during starvation-induced fruiting-body development in seven pairs of strains identical at traA. Collectively, our results suggest that most mechanisms of interference competition and inter-colony kin discrimination in natural populations of myxobacteria do not require OME. Finally, our experiments also indicate that several closely related laboratory reference strains kill some natural isolates by toxins delivered by a shared, OME-independent type VI secretion system (T6SS), suggesting that some antagonisms between sympatric natural isolates may also involve T6SS toxins.

sRNA-pathway genes regulating myxobacterial development exhibit clade-specific evolution.

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.

Divergence of functional effects among bacterial sRNA paralogs.

BACKGROUND: Non-coding small RNAs (sRNAs) regulate a variety of important biological processes across all life domains, including bacteria. However, little is known about the functional evolution of sRNAs in bacteria, which might occur via changes in sRNA structure and/or stability or changes in interactions between sRNAs and their associated regulatory networks, including target mRNAs. The sRNA Pxr functions as a developmental gatekeeper in the model cooperative bacterium Myxococcus xanthus. Specifically, Pxr prevents the initiation of fruiting body development when nutrients are abundant. Previous work has shown that Pxr appears to have a recent origin within a sub-clade of the myxobacteria, which allowed us to infer the most recent common ancestor of pxr and examine the divergence of Pxr since its origin. RESULTS: To test for inter-specific divergence in functional effects, extant pxr homologs from several species and their inferred ancestor were introduced into an M. xanthus deletion mutant lacking pxr. Both the inferred ancestral pxr and all extant alleles from species containing only one copy of pxr were found to control development in M. xanthus in a qualitatively similar manner to the native M. xanthus allele. However, multiple paralogs present in Cystobacter species exhibited divergent effects, with two paralogs controlling M. xanthus development but two others failing to do so. These differences may have occurred through changes in gene expression caused by apparent structural differences in the sRNA variants encoded by these paralogs. CONCLUSIONS: Taken together, our results suggest that Pxr plays a common fundamental role in developmental gene regulation across diverse species of myxobacteria but also that the functional effects of some Pxr variants may be evolving in some lineages.

A recent evolutionary origin of a bacterial small RNA that controls multicellular fruiting body development.

In animals and plants, non-coding small RNAs regulate the expression of many genes at the post-transcriptional level. Recently, many non-coding small RNAs (sRNAs) have also been found to regulate a variety of important biological processes in bacteria, including social traits, but little is known about the phylogenetic or mechanistic origins of such bacterial sRNAs. Here we propose a phylogenetic origin of the myxobacterial sRNA Pxr, which negatively regulates the initiation of fruiting body development in Myxococcus xanthus as a function of nutrient level, and also examine its diversification within the Myxococcocales order. Homologs of pxr were found throughout the Cystobacterineae suborder (with a few possible losses) but not outside this clade, suggesting a single origin of the Pxr regulatory system in the basal Cystobacterineae lineage. Rates of pxr sequence evolution varied greatly across Cystobacterineae sub-clades in a manner not predicted by overall genome divergence. A single copy of pxr was found in most species with 17% of nucleotide positions being polymorphic among them. However three tandem paralogs were present within the genus Cystobacter and these alleles together exhibited an elevated rate of divergence. There appears to have been strong selection for maintenance of a predicted stem-loop structure, as polymorphisms accumulated preferentially at loop or bulge regions or as complementary substitutions within predicted stems. All detected pxr homologs are located in the intergenic region between the σ(54)-dependent response regulator nla19 and a predicted NADH dehydrogenase gene, but other neighboring gene content has diversified.

Forespore signaling is necessary for pro-sigmaK processing during Bacillus subtilis sporulation despite the loss of SpoIVFA upon translational arrest.

The sigmaK checkpoint coordinates gene expression in the mother cell with signaling from the forespore during Bacillus subtilis sporulation. The signaling pathway involves SpoIVB, a serine peptidase produced in the forespore, which is believed to cross the innermost membrane surrounding the forespore and activate a complex of proteins, including BofA, SpoIVFA, and SpoIVFB, located in the outermost membrane surrounding the forespore. Activation of the complex allows proteolytic processing of pro-sigmaK, and the resulting sigmaK RNA polymerase transcribes genes in the mother cell. To investigate activation of the pro-sigmaK processing complex, the level of SpoIVFA in extracts of sporulating cells was examined by Western blot analysis. The SpoIVFA level decreased when pro-sigmaK processing began during sporulation. In extracts of a spoIVB mutant defective in forespore signaling, the SpoIVFA level failed to decrease normally and no processing of pro-sigmaK was observed. Although these results are consistent with a model in which SpoIVFA inhibits processing until the SpoIVB-mediated signal is received from the forespore, we discovered that loss of SpoIVFA was insufficient to allow processing under certain conditions, including static incubation of the culture and continued shaking after the addition of inhibitors of oxidative phosphorylation or translation. Under these conditions, loss of SpoIVFA was independent of spoIVB. The inability to process pro-sigmaK under these conditions was not due to loss of SpoIVFB, the putative processing enzyme, or to a requirement for ongoing synthesis of pro-sigmaK. Rather, it was found that the requirements for shaking of the culture, for oxidative phosphorylation, and for translation could be bypassed by mutations that uncouple processing from dependence on forespore signaling. This suggests that ongoing translation is normally required for efficient pro-sigmaK processing because synthesis of the SpoIVB signal protein is needed to activate the processing complex. When translation is blocked, synthesis of SpoIVB ceases, and the processing complex remains inactive despite the loss of SpoIVFA. Taken together, the results suggest that SpoIVB signaling activates the processing complex by performing another function in addition to causing loss of SpoIVFA or by causing loss of SpoIVFA in a different way than when translation is blocked. The results also demonstrate that the processing machinery can function in the absence of translation or an electrochemical gradient across membranes.