Microbial communities host many auxotrophs-organisms unable to synthesize one or more metabolites required for their growth. Auxotrophy is thought to confer an evolutionary advantage, yet auxotrophs ...must rely on other organisms that produce the metabolites they require. The mechanisms of metabolite provisioning by "producers" remain unknown. In particular, it is unclear how metabolites such as amino acids and cofactors, which are found inside the cell, are released by producers to become available to auxotrophs. Here, we explore metabolite secretion and cell lysis as two distinct possible mechanisms that result in the release of intracellular metabolites from producer cells. We measured the extent to which secretion or lysis of Escherichia coli and Bacteroides thetaiotaomicron amino acid producers can support the growth of engineered Escherichia coli amino acid auxotrophs. We found that cell-free supernatants and mechanically lysed cells provide minimal levels of amino acids to auxotrophs. In contrast, bacteriophage lysates of the same producer bacteria can support as many as 47 auxotroph cells per lysed producer cell. Each phage lysate released distinct levels of different amino acids, suggesting that in a microbial community the collective lysis of many different hosts by multiple phages could contribute to the availability of an array of intracellular metabolites for use by auxotrophs. Based on these results, we speculate that viral lysis could be a dominant mechanism of provisioning of intracellular metabolites that shapes microbial community structure.
All organisms rely on complex metabolites such as amino acids, nucleotides, and cofactors for essential metabolic processes. Some microbes synthesize these fundamental ingredients of life
, while ...others rely on uptake to fulfill their metabolic needs. Although certain metabolic processes are inherently "leaky," the mechanisms enabling stable metabolite provisioning among microbes in the absence of a host remain largely unclear. In particular, how can metabolite provisioning among free-living bacteria be maintained under the evolutionary pressure to economize resources? Salvaging, the process of "recycling and reusing," can be a metabolically efficient route to obtain access to required resources. Here, we show experimentally how precursor salvaging in engineered Escherichia coli populations can lead to stable, long-term metabolite provisioning. We find that salvaged cobamides (vitamin B
and related enzyme cofactors) are readily made available to nonproducing population members, yet salvagers are strongly protected from overexploitation. We also describe a previously unnoted benefit of precursor salvaging, namely, the removal of the nonfunctional, proliferation-inhibiting precursor. As long as compatible precursors are present, any microbe possessing the terminal steps of a biosynthetic process can, in principle, forgo
biosynthesis in favor of salvaging. Consequently, precursor salvaging likely represents a potent, yet overlooked, alternative to
biosynthesis for the acquisition and provisioning of metabolites in free-living bacterial populations.
Recycling gives new life to old things. Bacteria have the ability to recycle and reuse complex molecules they encounter in their environment to fulfill their basic metabolic needs in a resource-efficient way. By studying the salvaging (recycling and reusing) of vitamin B
precursors, we found that metabolite salvaging can benefit others and provide stability to a bacterial community at the same time. Salvagers of vitamin B
precursors freely share the result of their labor yet cannot be outcompeted by freeloaders, likely because salvagers retain preferential access to the salvaging products. Thus, salvaging may represent an effective, yet overlooked, mechanism of acquiring and provisioning nutrients in microbial populations.
Microbes commonly use metabolites produced by other organisms to compete effectively with others in their environment. As a result, microbial communities are composed of networks of metabolically ...interdependent organisms. How these networks evolve and shape population diversity, stability, and community function is a subject of active research. But how did these metabolic interactions develop initially? In particular, how and why are metabolites such as amino acids, cofactors, and nucleobases released for the benefit of others when there apparently is no incentive to do so? Here, we discuss the hypothesis that metabolite provisioning is not itself adaptive but rather can be a natural consequence of other evolved biological functions. We outline two examples of metabolite provisioning as a by-product of other functions by considering cell lysis and regulated metabolite efflux outside their canonical roles and explore their potential to facilitate the emergence of interdependent metabolite sharing.
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Promoting axon growth after peripheral nerve injury may support recovery. Soluble laminin polymers formed at pH 4 (aLam) accelerate axon growth from adult dorsal root ganglion neurons ...in vitro. We used an adult rat model of a peripheral (peroneal) nerve crush to investigate whether an injection of aLam enhances axon growth and functional recovery in vivo. Rats that received an injection of aLam into the crush at 2 days post-injury show significant improvements in hind limb motor function at 2 and 5 weeks after injury compared with control rats that received phosphate-buffered saline. Functional improvement was not associated with changes in sensitivity to thermal or mechanical stimuli. Treatment with aLam decreased the occurrence of autophagia and abolished non-compliance with treadmill walking. Rats treated with aLam showed increased axon presence in the crush site at 2 weeks post-injury and larger axon diameter at 10 weeks post-injury compared with controls. Treatment with aLam did not affect Schwann cell presence or axon myelination. Our results demonstrated that aLam accelerates axon growth and maturity in a crushed peroneal nerve associated with expedited hind limb motor function recovery. Our data support the therapeutic potential of injectable aLam polymers for treatment of peripheral nerve crush injuries.
Incidence of peripheral nerve injury has been estimated to be as high as 5% of all cases entering a Level 1 trauma center and the majority of cases are young males. Peripheral nerves have some endogenous repair capabilities, but overall recovery of function remains limited, which typically has devastating effects on the individual, family, and society, as wages are lost and rehabilitation is extended until the nerves can repair. We report here that laminin polymers injected into a crush accelerated repair and recovery, had no adverse effects on sensory function, obliterated non-compliance for walking tests, and decreased the occurrence of autophagia. These data support the use of laminin polymers for safe and effective recovery after peripheral nerve injury.
Alternative splicing (AS) is a robust generator of mammalian transcriptome complexity. Splice site specification is controlled by interactions of cis-acting determinants on a transcript with specific ...RNA binding proteins. These interactions are frequently localized to the intronic U-rich polypyrimidine tracts (PPT) located 5' to the majority of splice acceptor junctions. αCPs (also referred to as polyC-binding proteins (PCBPs) and hnRNPEs) comprise a subset of KH-domain proteins with high affinity and specificity for C-rich polypyrimidine motifs. Here, we demonstrate that αCPs promote the splicing of a defined subset of cassette exons via binding to a C-rich subset of polypyrimidine tracts located 5' to the αCP-enhanced exonic segments. This enhancement of splice acceptor activity is linked to interactions of αCPs with the U2 snRNP complex and may be mediated by cooperative interactions with the canonical polypyrimidine tract binding protein, U2AF65. Analysis of αCP-targeted exons predicts a substantial impact on fundamental cell functions. These findings lead us to conclude that the αCPs play a direct and global role in modulating the splicing activity and inclusion of an array of cassette exons, thus driving a novel pathway of splice site regulation within the mammalian transcriptome.
Microbes exist in complex, multispecies communities where they participate in nutritional interactions that shape microbial community structure, function and stability. Nutritional interactions can ...range from competition for shared nutrients, to mutualisms, where nutrients are reciprocally exchanged in ways that benefit both partners. Not all microbes are able to produce all of their required nutrients, and as a result, rely on receiving nutrients from neighboring microbes. I am interested in understanding mechanisms of nutrient release and its implications in supporting microbial communities.In Chapter 1, I provide background on how microbes interact in communities, and discuss examples of different types of microbial interactions. I then review how microbes impact their surroundings by releasing by-products and metabolites into their environment, that can then be used by other microbes as nutrients. Next, I explore cooperative and mutualistic behaviors, and theories for how we think cooperative interactions evolve overtime, before discussing auxotrophy, and how this trait may have evolved via adaptive evolution. I end Chapter 1 by discussing cell death, different forms of cell lysis, including bacteriophage-mediated lysis, and review what is known about how these processes are related to nutrient release and nutrient cycling in microbial communities.In Chapter 2, I discuss two theories for how I think metabolites can be released into the environment to facilitate interdependent metabolite sharing. Microbial communities are composed of complex networks of metabolically interdependent organisms. But it is unclear how these nutritional networks evolve. In particular, the incentive for releasing metabolites, such as amino acids, vitamins and nucleobases is not obvious. I discuss that nutrient release could be a by-product of processes, like cell lysis and regulated metabolite efflux, that could facilitate the emergence of interdependent metabolite sharing.I experimentally test these predictions in Chapter 3 by hypothesizing that bacteriophage-mediated lysis is a dominant mechanism of nutrient release that can support amino acid auxotrophs. I use bacterial growth assays to investigate how well supernatants, mechanical cell lysates, and phage-generated lysates are able to support a set of amino acid auxotrophs. I found that supernatants and mechanical lysates minimally support auxotrophs, and phage lysates release a significant amount of bioavailable nutrients, suggesting that in nature, phage are likely to play a large role in providing auxotrophs with their required nutrient.Chapter 4 explores secretion as a mechanism of nutrient provisioning. More specifically, I test how nutrient overproduction can occur as result of auxotrophic mutations, and the implications of this nutrient overproduction on co-culture growth. To test this I developed an obligate mutualistic synthetic co-culture using two engineered E. coli that reciprocally exchange vitamin B12 and methionine. I show that co-culture growth is limited by methionine secretion, and reveal how specific auxotrophic mutations are able to increase flux through the methionine biosynthesis pathway to improve co-culture growth.In addition to exploring mechanisms of nutrient provisioning, I also conducted a sociological project about doctoral students in the biological sciences. In Chapter 5, I investigate key elements of socialization that doctoral students in the biological sciences experience as they navigate their graduate programs. From interviews with over 30 doctoral students, I highlight how informal interactions affect students access to scientific help and expertise.
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