Mutation of rpoB Shifts the Nutrient Threshold Triggering Myxococcus Multicellular Development.

The ability to perceive and respond to environmental change is essential to all organisms. In response to nutrient depletion, cells of the soil-dwelling δ-proteobacterium Myxococcus xanthus undergo collective morphogenesis into multicellular fruiting bodies and transform into stress-resistant spores. This process is strictly regulated by gene networks that incorporate both inter- and intracellular signals. While commonly studied M. xanthus reference strains and some natural isolates undergo development only in nutrient-poor conditions, some lab mutants and other natural isolates commit to development at much higher nutrient levels, but mechanisms enabling such rich medium development remain elusive. Here we investigate the genetic basis of rich medium development in one mutant and find that a single amino acid change (S534L) in RpoB, the ß-subunit of RNA polymerase, is responsible for the phenotype. Ectopic expression of the mutant rpoB allele was sufficient to induce nutrient-rich development. These results suggest that the universal bacterial transcription machinery bearing the altered ß-subunit can relax regulation of developmental genes that are normally strictly controlled by the bacterial stringent response. Moreover, the mutation also pleiotropically mediates a tradeoff in fitness during vegetative growth between high vs. low nutrient conditions and generates resistance to exploitation by a developmental cheater. Our findings reveal a previously unknown connection between the universal transcription machinery and one of the most behaviorally complex responses to environmental stress found among bacteria.

Bacterial predator-prey coevolution accelerates genome evolution and selects on virulence-associated prey defences.

Generalist bacterial predators are likely to strongly shape many important ecological and evolutionary features of microbial communities, for example by altering the character and pace of molecular evolution, but investigations of such effects are scarce. Here we report how predator-prey interactions alter the evolution of fitness, genomes and phenotypic diversity in coevolving bacterial communities composed of Myxococcus xanthus as predator and Escherichia coli as prey, relative to single-species controls. We show evidence of reciprocal adaptation and demonstrate accelerated genomic evolution specific to coevolving communities, including the rapid appearance of mutator genotypes. Strong parallel evolution unique to the predator-prey communities occurs in both parties, with predators driving adaptation at two prey traits associated with virulence in bacterial pathogens-mucoidy and the outer-membrane protease OmpT. Our results suggest that generalist predatory bacteria are important determinants of how complex microbial communities and their interaction networks evolve in natural habitats.

Kin discrimination and outer membrane exchange in Myxococcus xanthus: A comparative analysis among natural isolates.

Genetically similar cells of the soil bacterium Myxococcus xanthus cooperate at multiple social behaviours, including motility and multicellular development. Another social interaction in this species is outer membrane exchange (OME), a behaviour of unknown primary benefit in which cells displaying closely related variants of the outer membrane protein TraA transiently fuse and exchange membrane contents. Functionally incompatible TraA variants do not mediate OME, which led to the proposal that TraA incompatibilities determine patterns of intercellular cooperation in nature, but how this might occur remains unclear. Using natural isolates from a centimetre-scale patch of soil, we analyse patterns of TraA diversity and ask whether relatedness at TraA is causally related to patterns of kin discrimination in the form of both colony-merger incompatibilities (CMIs) and interstrain antagonisms. A large proportion of TraA functional diversity documented among global isolates is predicted to be contained within this cm-scale population. We find evidence of balancing selection on the highly variable PA14-portion of TraA and extensive transfer of traA alleles across genomic backgrounds. CMIs are shown to be common among strains identical at TraA, suggesting that CMIs are not generally caused by TraA dissimilarity. Finally, it has been proposed that interstrain antagonisms might be caused by OME-mediated toxin transfer. However, we predict that most strain pairs previously shown to exhibit strong antagonisms are incapable of OME due to TraA dissimilarity. Overall, our results suggest that most documented patterns of kin discrimination in a natural population of M. xanthus are not causally related to the TraA sequences of interactants.

A conserved stem of the Myxococcus xanthus sRNA Pxr controls sRNA accumulation and multicellular development.

The small RNA (sRNA) Pxr negatively controls multicellular fruiting body formation in the bacterium Myxococcus xanthus, inhibiting the transition from growth to development when nutrients are abundant. Like many other prokaryotic sRNAs, Pxr is predicted to fold into three stem loops (SL1-SL3). SL1 and SL2 are highly conserved across the myxobacteria, whereas SL3 is much more variable. SL1 is necessary for the regulatory function of Pxr but the importance of SL3 in this regard is unknown. To test for cis genetic elements required for Pxr function, we deleted the entire pxr gene from a developmentally defective strain that fails to remove Pxr-mediated blockage of development and reintroduced variably truncated fragments of the pxr region to test for their ability to block development. These truncations demonstrated that SL3 is necessary for Pxr function in the defective strain. We further show that a highly conserved eight-base-pair segment of SL3 is not only necessary for Pxr to block development in the defective strain under starvation conditions, but is also required for Pxr to prevent fruiting body development by a developmentally proficient wild-type strain under high-nutrient conditions. This conserved segment of SL3 is also necessary for detectable levels of Pxr to accumulate, suggesting that this segment either stabilizes Pxr against premature degradation during vegetative growth or positively regulates its transcription.

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

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 σ54 response regulator (RR) gene MXAN_1078 (named pxrR here), immediately upstream of pxr PxrR was found to positively regulate pxr transcription, presumably via the σ54 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. IMPORTANCE: Many 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 σ54 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.

A shift from magnitude to sign epistasis during adaptive evolution of a bacterial social trait.

Although the importance of epistasis in evolution has long been recognized, remarkably little is known about the processes by which epistatic interactions evolve in real time in specific biological systems. Here, we have characterized how the epistatic fitness relationship between a social gene and an adapting genome changes radically over a short evolutionary time frame in the social bacterium Myxococcus xanthus. We show that a highly beneficial effect of this social gene in the ancestral genome is gradually reduced--and ultimately reversed into a deleterious effect--over the course of an experimental adaptive trajectory in which a primitive form of novel cooperation evolved. This reduction and reversal of a positive social allelic effect is driven solely by changes in the genetic context in which the gene is expressed as new mutations are sequentially fixed during adaptive evolution, and explicitly demonstrates a significant evolutionary change in the genetic architecture of an ecologically important social trait.

Adaptive evolution of an sRNA that controls Myxococcus development.

Small RNA (sRNA) molecules regulate a vast array of processes in biology, but evidence for adaptive evolution of sRNA sequences has been indirect. Here, we identify an sRNA, Pxr, that negatively regulates fruiting body development in Myxococcus xanthus. We further show that a spontaneous evolutionary mutation in Pxr abolished its regulatory function and thereby adaptively restored developmental proficiency to a socially defective M. xanthus cheater. In wild-type M. xanthus, development is initiated only upon starvation, but deletion of pxr allows development to proceed even while nutrients remain abundant. Thus, Pxr serves as a major checkpoint controlling the transition from growth to development in the myxobacteria. These findings show that an sRNA molecule governs a complex form of multicellular development in prokaryotes and directly demonstrate the ability of sRNA regulators to facilitate evolutionary adaptations of major phenotypic effect.

Evolution of an obligate social cheater to a superior cooperator.

Obligate relationships have evolved many times and can be parasitic or mutualistic. Obligate organisms rely on others to survive and thus coevolve with their host or partner. An important but little explored question is whether obligate status is an evolutionarily terminal condition or whether obligate lineages can evolve back to an autonomous lifestyle. The bacterium Myxococcus xanthus survives starvation by the social development of spore-bearing fruiting bodies. Some M. xanthus genotypes defective at fruiting body development in isolation can nonetheless exploit proficient genotypes in chimaeric groups. Here we report an evolutionary transition from obligate dependence on an altruistic host to an autonomous mode of social cooperation. This restoration of social independence was caused by a single mutation of large effect that confers fitness superiority over both ancestral genotypes, including immunity from exploitation by the ancestral cheater. Thus, a temporary state of obligate cheating served as an evolutionary stepping-stone to a novel state of autonomous social dominance.

Evolution of novel cooperative swarming in the bacterium Myxococcus xanthus.

Cooperation among individuals is necessary for evolutionary transitions to higher levels of biological organization. In such transitions, groups of individuals at one level (such as single cells) cooperate to form selective units at a higher level (such as multicellular organisms). Though the evolution of cooperation is difficult to observe directly in higher eukaryotes, microorganisms do offer such an opportunity. Here we report the evolution of novel cooperative behaviour in experimental lineages of the bacterium Myxococcus xanthus. Wild-type strains of M. xanthus exhibit socially dependent swarming across soft surfaces by a mechanism known as 'S-motility' that requires the presence of extracellular type IV pili. In lineages of M. xanthus unable to make pili, a new mechanistic basis for cooperative swarming evolved. Evolved swarming is mediated, at least in part, by enhanced production of an extracellular fibril matrix that binds cells-and their evolutionary interests-together. Though costly to individuals, fibril production greatly enhanced population expansion in groups of interconnected cells. These results show that fundamental transitions to primitive cooperation can readily occur in bacteria.