Hyphal compartmentalization and sporulation in Streptomyces require the conserved cell division protein SepX

Filamentous actinobacteria such as Streptomyces undergo two distinct modes of cell division, leading to partitioning of growing hyphae into multicellular compartments via cross-walls, and to septation and release of unicellular spores. Specific determinants for cross-wall formation and the importance of hyphal compartmentalization for Streptomyces development are largely unknown. Here we show that SepX, an actinobacterial-specific protein, is crucial for both cell division modes in Streptomyces venezuelae. Importantly, we find that sepX-deficient mutants grow without cross-walls and that this substantially impairs the fitness of colonies and the coordinated progression through the developmental life cycle. Protein interaction studies and live-cell imaging suggest that SepX contributes to the stabilization of the divisome, a mechanism that also requires the dynamin-like protein DynB. Thus, our work identifies an important determinant for cell division in Streptomyces that is required for cellular development and sporulation.

So What? Who Cares?

In Chapter 9, the closing chapter, the authors raise four “So what?” issues that our fellow learners and aspiring health system designers should address: getting good value for money while simultaneously satisfying individual consumers of care and guaranteeing that all citizens can access the health care their nation provides; understanding the system’s developmental life cycle and capitalizing on a zone of innovation available in existing health system design; comprehending what reconceptualization means and giving some examples of what it might look like; and appreciating that a high-value health system needs to be owned by all and requires each and every citizen to do their part in preserving, protecting, and sustaining it.

Multicellular growth and sporulation in filamentous actinobacteria require the conserved cell division protein SepX

Filamentous actinobacteria like Streptomyces undergo two distinct modes of cell division, leading to the partitioning of growing hyphae into multicellular compartments via cross-walls and to the septation and release of unicellular spores. While some progress has been made towards the regulation of sporulation-specific cell division, specific determinants for cross-wall formation and the importance of hyphal compartmentalization for Streptomyces development have remained unknown. Here we describe SepX, an actinobacterial-specific protein that is crucial for both cell division events in Streptomyces. We show that sepX-deficient mutants grow without cross-walls and that this substantially impairs the fitness of colonies and the coordinated progression through the developmental life cycle. Protein interaction studies and live-cell imaging suggest that SepX functions to spatially stabilize the divisome, a mechanism that also requires the dynamin-like protein DynB. Collectively, this work identifies an important determinant for cell division in filamentous actinobacteria that is required for multicellular development and sporulation.

Healing journey: a qualitative analysis of the healing experiences of Americans suffering from trauma and illness

ObjectivesTo elucidate pathways to healing for people having suffered injury to the integrity of their function as a human being.MethodsA team of physician-analysts conducted thematic analyses of in-depth interviews of 23 patients who experienced healing, as identified by six primary care physicians purposefully selected as exemplary healers.ResultsPeople in the sample experienced healing journeys that spanned a spectrum from overcoming unspeakable trauma and then becoming healers themselves to everyday heroes functioning well despite ongoing serious health challenges.The degree and quality of suffering experienced by each individual is framed by contextual factors that include personal characteristics, timing of their initial or ongoing wounding in the developmental life cycle and prior and current relationships.In the healing journey, bridges from suffering are developed to healing resources/skills and connections to helpers outside themselves. These bridges often evolve in fits and starts and involve persistence and developing a sense of safety and trust.From the iteration between suffering and developing resources and connections, a new state emerges that involves hope, self-acceptance and helping others. Over time, this leads to healing that includes a sense of integrity and flourishing in the pursuit of meaningful goals and purpose.ConclusionMoving from being wounded, through suffering to healing, is possible. It is facilitated by developing safe, trusting relationships and by positive reframing that moves through the weight of responsibility to the ability to respond.

CpG Oligodeoxynucleotide Treatment Enhances Innate Resistance and Acquired Immunity to African Trypanosomes

ABSTRACTRelative resistance to African trypanosomiasis is based on the development of a type I cytokine response, which is partially dependent on innate immune responses generated through MyD88 and Toll-like receptor 9 (TLR9). Therefore, we asked whether enhancement of the immune response by artificial stimulation with CpG oligodeoxynucleotide (ODN), a TLR9 agonist, would result in enhanced protection against trypanosomes. In susceptible BALB/c mice, relative resistance to infection was significantly enhanced by CpG ODN treatment and was associated with decreased parasite burden, increased cytokine production, and elevated parasite-specific B- and T-cell responses. In relatively resistant C57BL/6 mice, survival was not enhanced but early parasitemia levels were reduced 100-fold and the majority of the parasites were nondividing, short stumpy (SS) forms. CpG ODN treatment of lymphocyte-deficient C57BL/6-scidand BALB/cByJ-scidmice also enhanced survival and reduced parasitemia, indicating that innate resistance to trypanosome infection can be enhanced. In C57BL/6-scidand BALB/cByJ-scidmice, the parasites were also predominantly SS forms during the outgrowth of parasitemia. However, the effect of CpG ODN treatment on parasite morphology was not as marked in gamma interferon (IFN-γ)-knockout mice, suggesting that downstream effects of IFN-γ production may play a discrete role in parasite cell differentiation. Overall, these studies provide the first evidence that enhancement of resistance to African trypanosomes can be induced in susceptible animals in a TLR9-dependent manner and that CpG ODN treatment may influence the developmental life cycle of the parasites.

Integration into the Phage Attachment Site, attB, Impairs Multicellular Differentiation in Stigmatella aurantiaca

ABSTRACT Stigmatella aurantiaca displays a complex developmental life cycle in response to starvation conditions that results in the formation of tree-like fruiting bodies capable of producing spores. The phage Mx8, first isolated from the close relative Myxococcus xanthus, is unable to infect S. aurantiaca cells and integrate into the genome. However, plasmids containing Mx8 fragments encoding the integrase and attP are able to integrate at the attB locus in the S. aurantiaca genome by site-specific recombination. After recombination between attP and attB, the S. aurantiaca cells were incapable of building normal fruiting bodies but formed clumps and fungus-like structures characteristic of intermediate stages of development displayed by the wild type. We identified two tRNA genes, trnD and trnV, encoding tRNAAsp and tRNAVal, respectively, composing an operon at the attB locus of S. aurantiaca. Integration of attP-containing plasmids resulted in the incorporation of the tMx8 terminator sequence, in addition to a short sequence of Mx8 DNA downstream of trnD. The integrant was unable to process the trnD transcript at the normal 3′ processing site and displayed a lower level of expression of the trnVD operon. In addition, several developmentally regulated proteins were no longer produced in mutants following insertion at the attB locus. We hypothesize that the integration of the tMx8 terminator sequence results in reduced levels of mature tRNAAsp and tRNAVal and that altered protein production during development is thereby responsible for the observed phenotype. The trnVD locus thus defines a new developmental checkpoint for Stigmatella aurantiaca.

Phenotypic differentiation and seeding dispersal in non-mucoid and mucoid Pseudomonas aeruginosa biofilms

There is growing evidence that Pseudomonas aeruginosa biofilms exhibit a multicellular developmental life cycle analogous to that of the myxobacteria. In non-mucoid PAO1 biofilms cultured in glass flow cells the phenotypic differentiation of microcolonies into a motile phenotype in the interior of the microcolony and a non-motile surrounding ‘wall phenotype’ are described. After differentiation the interior cells coordinately evacuated the microcolony from local break out points and spread over the wall of the flow cell, suggesting that the specialized microcolonies were analogous to crude fruiting bodies. A microcolony diameter of approximately 80 μm was required for differentiation, suggesting that regulation was related to cell density and mass transfer conditions. This phenomenon was termed ‘seeding dispersal’ to differentiate it from ‘erosion’ which is the passive removal of single cells by fluid shear. Using the flow cell culturing method, in which reproducible seeding phenotype in PAO1 wild-type was demonstrated, the effects of quorum sensing (QS) and rhamnolipid production (factors previously identified as important in determining biofilm structure) on seeding dispersal using knockout mutants isogenic with PAO1 was investigated. Rhamnolipid (rhlA) was not required for seeding dispersal but las/rhl QS (PAO1-JP2) was, in our system. To assess the clinical relevance of these data, mucoid P. aeruginosa cystic fibrosis isolate FRD1 was also investigated and was seeding-dispersal-negative.