Barcoding Populations of Pseudomonas fluorescens SBW25.

In recent years, evolutionary biologists have developed an increasing interest in the use of barcoding strategies to study eco-evolutionary dynamics of lineages within evolving populations and communities. Although barcoded populations can deliver unprecedented insight into evolutionary change, barcoding microbes presents specific technical challenges. Here, strategies are described for barcoding populations of the model bacterium Pseudomonas fluorescens SBW25, including the design and cloning of barcoded regions, preparation of libraries for amplicon sequencing, and quantification of resulting barcoded lineages. In so doing, we hope to aid the design and implementation of barcoding methodologies in a broad range of model and non-model organisms.

A mechanism for the initiation of allergen-induced T helper type 2 responses.

Both metazoan parasites and simple protein allergens induce T helper type 2 (TH2) immune responses, but the mechanisms by which the innate immune system senses these stimuli are unknown. In addition, the cellular source of cytokines that control TH2 differentiation in vivo has not been defined. Here we showed that basophils were activated and recruited to the draining lymph nodes specifically in response to TH2-inducing allergen challenge. Furthermore, we demonstrate that the basophil was the accessory cell type required for TH2 induction in response to protease allergens. Finally, we show that basophils were directly activated by protease allergens and produced TH2-inducing cytokines, including interleukin 4 and thymic stromal lymphopoietin, which are involved in TH2 differentiation in vivo.

Lack of Foxp3 function and expression in the thymic epithelium.

Foxp3 is essential for the commitment of differentiating thymocytes to the regulatory CD4(+) T (T reg) cell lineage. In humans and mice with a genetic Foxp3 deficiency, absence of this critical T reg cell population was suggested to be responsible for the severe autoimmune lesions. Recently, it has been proposed that in addition to T reg cells, Foxp3 is also expressed in thymic epithelial cells where it is involved in regulation of early thymocyte differentiation and is required to prevent autoimmunity. Here, we used genetic tools to demonstrate that the thymic epithelium does not express Foxp3. Furthermore, we formally showed that genetic abatement of Foxp3 in the hematopoietic compartment, i.e. in T cells, is both necessary and sufficient to induce the autoimmune lesions associated with Foxp3 loss. In contrast, deletion of a conditional Foxp3 allele in thymic epithelial cells did not result in detectable changes in thymocyte differentiation or pathology. Therefore, in mice the only known role for Foxp3 remains promotion of T reg cell differentiation within the T cell lineage, whereas there is no role for Foxp3 in thymic epithelial cells.

The genome sequence of the Large Brook Dun, Ecdyonurus torrentis (Kimmins, 1942).

We present a genome assembly from an individual female Ecdyonurus torrentis (the Large Brook Dun; Arthropoda; Insecta; Ephemeroptera; Heptageniidae). The genome sequence is 503.2 megabases in span. Most of the assembly is scaffolded into 11 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 15.69 kilobases in length.

The genome sequence of a stonefly, Nemoura dubitans (Morton, 1894).

We present a genome assembly from an individual female Nemoura dubitans (a stonefly; Arthropoda; Insecta; Plecoptera; Nemouridae). The genome sequence is 321.0 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 15.73 kilobases in length.

The genome sequence of the Northern Summer Mayfly, Siphlonurus alternatus (Say, 1824).

We present a genome assembly from an individual male Siphlonurus alternatus (the Northern Summer Mayfly; Arthropoda; Insecta; Ephemeroptera; Siphlonuridae). The genome sequence is 455.8 megabases in span. Most of the assembly is scaffolded into 11 chromosomal pseudomolecules, including the X sex chromosome. The mitochondrial genome has also been assembled and is 19.36 kilobases in length.

Population dynamics of cross-protection against ß-lactam antibiotics in droplet microreactors.

Introduction: Bacterial strains that are resistant to antibiotics may protect not only themselves, but also sensitive bacteria nearby if resistance involves antibiotic degradation. Such cross-protection poses a challenge to effective antibiotic therapy by enhancing the long-term survival of bacterial infections, however, the current understanding is limited. Methods: In this study, we utilize an automated nanoliter droplet analyzer to study the interactions between Escherichia coli strains expressing a ß-lactamase (resistant) and those not expressing it (sensitive) when exposed to the ß-lactam antibiotic cefotaxime (CTX), with the aim to define criteria contributing to cross-protection. Results: We observed a cross-protection window of CTX concentrations for the sensitive strain, extending up to approximately 100 times its minimal inhibitory concentration (MIC). Through both microscopy and enzyme activity analyses, we demonstrate that bacterial filaments, triggered by antibiotic stress, contribute to cross-protection. Discussion: The antibiotic concentration window for cross-protection depends on the difference in ß-lactamase activity between co-cultured strains: larger differences shift the 'cross-protection window' toward higher CTX concentrations. Our findings highlight the dependence of opportunities for cross-protection on the relative resistance levels of the strains involved and suggest a possible specific role for filamentation.

Identifying and tracking mobile elements in evolving compost communities yields insights into the nanobiome.

Microbial evolution is driven by rapid changes in gene content mediated by horizontal gene transfer (HGT). While mobile genetic elements (MGEs) are important drivers of gene flux, the nanobiome-the zoo of Darwinian replicators that depend on microbial hosts-remains poorly characterised. New approaches are necessary to increase our understanding beyond MGEs shaping individual populations, towards their impacts on complex microbial communities. A bioinformatic pipeline (xenoseq) was developed to cross-compare metagenomic samples from microbial consortia evolving in parallel, aimed at identifying MGE dissemination, which was applied to compost communities which underwent periodic mixing of MGEs. We show that xenoseq can distinguish movement of MGEs from demographic changes in community composition that otherwise confounds identification, and furthermore demonstrate the discovery of various unexpected entities. Of particular interest was a nanobacterium of the candidate phylum radiation (CPR) which is closely related to a species identified in groundwater ecosystems (Candidatus Saccharibacterium), and appears to have a parasitic lifestyle. We also highlight another prolific mobile element, a 313 kb plasmid hosted by a Cellvibrio lineage. The host was predicted to be capable of nitrogen fixation, and acquisition of the plasmid coincides with increased ammonia production. Taken together, our data show that new experimental strategies combined with bioinformatic analyses of metagenomic data stand to provide insight into the nanobiome as a driver of microbial community evolution.

Minimal Surviving Inoculum in Collective Antibiotic Resistance.

A common strategy used by bacteria to resist antibiotics is enzymatic degradation or modification. This reduces the antibiotic threat in the environment and is therefore potentially a collective mechanism that also enhances the survival of nearby cells. Collective resistance is of clinical significance, yet a quantitative understanding at the population level is still incomplete. Here, we develop a general theoretical framework of collective resistance by antibiotic degradation. Our modeling study reveals that population survival crucially depends on the ratio of timescales of two processes: the rates of population death and antibiotic removal. However, it is insensitive to molecular, biological, and kinetic details of the underlying processes that give rise to these timescales. Another important aspect of antibiotic degradation is the degree of cooperativity, related to the permeability of the cell wall to antibiotics and enzymes. These observations motivate a coarse-grained, phenomenological model, with two compound parameters representing the population's race to survival and single-cell effective resistance. We propose a simple experimental assay to measure the dose-dependent minimal surviving inoculum and apply it to Escherichia coli expressing several types of ß-lactamase. Experimental data analyzed within the theoretical framework corroborate it with good agreement. Our simple model may serve as a reference for more complex situations, such as heterogeneous bacterial communities. IMPORTANCE Collective resistance occurs when bacteria work together to decrease the concentration of antibiotics in their environment, for example, by actively breaking down or modifying them. This can help bacteria survive by reducing the effective antibiotic concentration below their threshold for growth. In this study, we used mathematical modeling to examine the factors that influence collective resistance and to develop a framework to understand the minimum population size needed to survive a given initial antibiotic concentration. Our work helps to identify generic mechanism-independent parameters that can be derived from population data and identifies combinations of parameters that play a role in collective resistance. Specifically, it highlights the relative timescales involved in the survival of populations that inactivate antibiotics, as well as the levels of cooperation versus privatization. The results of this study contribute to our understanding of population-level effects on antibiotic resistance and may inform the design of antibiotic therapies.

The Fitness of Beta-Lactamase Mutants Depends Nonlinearly on Resistance Level at Sublethal Antibiotic Concentrations.

Adaptive evolutionary processes are constrained by the availability of mutations which cause a fitness benefit and together make up the fitness landscape, which maps genotype space onto fitness under specified conditions. Experimentally derived fitness landscapes have demonstrated a predictability to evolution by identifying limited "mutational routes" that evolution by natural selection may take between low and high-fitness genotypes. However, such studies often utilize indirect measures to determine fitness. We estimated the competitive fitness of mutants relative to all single-mutation neighbors to describe the fitness landscape of three mutations in a ß-lactamase enzyme. Fitness assays were performed at sublethal concentrations of the antibiotic cefotaxime in a structured and unstructured environment. In the unstructured environment, the antibiotic selected for higher-resistance types-but with an equivalent fitness for a subset of mutants, despite substantial variation in resistance-resulting in a stratified fitness landscape. In contrast, in a structured environment with a low antibiotic concentration, antibiotic-susceptible genotypes had a relative fitness advantage, which was associated with antibiotic-induced filamentation. These results cast doubt that highly resistant genotypes have a unique selective advantage in environments with subinhibitory concentrations of antibiotics and demonstrate that direct fitness measures are required for meaningful predictions of the accessibility of evolutionary routes. IMPORTANCE The evolution of antibiotic-resistant bacterial populations underpins the ongoing antibiotic resistance crisis. We aim to understand how antibiotic-degrading enzymes can evolve to cause increased resistance, how this process is constrained, and whether it can be predictable. To this end, competition experiments were performed with a combinatorially complete set of mutants of a ß-lactamase gene subject to subinhibitory concentrations of the antibiotic cefotaxime. While some mutations confer on their hosts high resistance to cefotaxime, in competition these mutations do not always confer a selective advantage. Specifically, high-resistance mutants had equivalent fitnesses despite different resistance levels and even had selective disadvantages under conditions involving spatial structure. Together, our findings suggest that the relationship between resistance level and fitness at subinhibitory concentrations is complex; predicting the evolution of antibiotic resistance requires knowledge of the conditions that select for resistant genotypes and the selective advantage evolved types have over their predecessors.

Lymphatic endothelial murine chloride channel calcium-activated 1 is a ligand for leukocyte LFA-1 and Mac-1.

The lymphatic circulation mediates drainage of fluid and cells from the periphery through lymph nodes, facilitating immune detection of lymph-borne foreign Ags. The 10.1.1 mAb recognizes a lymphatic endothelial Ag, in this study purified by Ab-affinity chromatography. SDS-PAGE and mass spectrometry identified murine chloride channel calcium-activated 1 (mCLCA1) as the 10.1.1 Ag, a 90-kDa cell-surface protein expressed in lymphatic endothelium and stromal cells of spleen and thymus. The 10.1.1 Ab-affinity chromatography also purified LFA-1, an integrin that mediates leukocyte adhesion to endothelium. This mCLCA1-LFA-1 interaction has functional consequences, as lymphocyte adhesion to lymphatic endothelium was blocked by 10.1.1 Ab bound to endotheliumor by LFA-1 Ab bound to lymphocytes. Lymphocyte adhesion was increased by cytokine treatment of lymphatic endothelium in association with increased expression of ICAM-1, an endothelial surface protein that is also a ligand for LFA-1. By contrast, mCLCA1 expression and the relative contribution of mCLCA1 to lymphocyte adhesion were unaffected by cytokine activation, demonstrating that mCLCA1 and ICAM-1 interactions with LFA-1 are differentially regulated. mCLCA1 also bound to the LFA-1-related Mac-1 integrin that is preferentially expressed on leukocytes. mCLCA1-mediated adhesion of Mac-1- or LFA-1-expressing leukocytes to lymphatic vessels and lymph node lymphatic sinuses provides a target for investigation of lymphatic involvement in leukocyte adhesion and trafficking during the immune response.

Polymer/boron nitride nanocomposite materials for superior thermal transport performance.

Boron nitride nanosheets were dispersed in polymers to give composite films with excellent thermal transport performances approaching the record values found in polymer/graphene nanocomposites. Similarly high performance at lower BN loadings was achieved by aligning the nanosheets in poly(vinyl alcohol) matrix by simple mechanical stretching (see picture).

Contrasting models of promiscuous gene expression by thymic epithelium.

Medullary thymic epithelial cells (mTECs) express a broad spectrum of tissue- restricted self-antigens (TRAs), which are required for the development of central tolerance. A new study suggests that TRA expression is a specialized property of terminally differentiated mTECs. However, as discussed here, an alternative model-whereby TRA expression is regulated by conserved developmental programs active in developing mTECs-may be equally plausible.

Developmental regulation of Foxp3 expression during ontogeny.

Thymectomy of neonatal mice can result in the development of autoimmune pathology. It has been proposed that thymic output of regulatory T (T reg) cells is delayed during ontogeny and that the development of autoimmune disease in neonatally thymectomized mice is caused by the escape of self-reactive T cells before thymectomy without accompanying T reg cells. However, the kinetics of T reg cell production within the thymus during ontogeny has not been assessed. We demonstrate that the development of Foxp3-expressing T reg cells is substantially delayed relative to nonregulatory thymocytes during ontogeny. Based on our data, we speculate that induction of Foxp3 in developing thymocytes and, thus, commitment to the T reg cell lineage is facilitated by a signal largely associated with the thymic medulla.

Notch-deficient skin induces a lethal systemic B-lymphoproliferative disorder by secreting TSLP, a sentinel for epidermal integrity.

Epidermal keratinocytes form a highly organized stratified epithelium and sustain a competent barrier function together with dermal and hematopoietic cells. The Notch signaling pathway is a critical regulator of epidermal integrity. Here, we show that keratinocyte-specific deletion of total Notch signaling triggered a severe systemic B-lymphoproliferative disorder, causing death. RBP-j is the DNA binding partner of Notch, but both RBP-j-dependent and independent Notch signaling were necessary for proper epidermal differentiation and lipid deposition. Loss of both pathways caused a persistent defect in skin differentiation/barrier formation. In response, high levels of thymic stromal lymphopoietin (TSLP) were released into systemic circulation by Notch-deficient keratinocytes that failed to differentiate, starting in utero. Exposure to high TSLP levels during neonatal hematopoiesis resulted in drastic expansion of peripheral pre- and immature B-lymphocytes, causing B-lymphoproliferative disorder associated with major organ infiltration and subsequent death, a previously unappreciated systemic effect of TSLP. These observations demonstrate that local skin perturbations can drive a lethal systemic disease and have important implications for a wide range of humoral and autoimmune diseases with skin manifestations.

Lessons from thymic epithelial heterogeneity: FoxN1 and tissue-restricted gene expression by extrathymic, endodermally derived epithelium.

Modeling of thymic epithelial differentiation has been guided by several important underlying assumptions. One is that within epithelial tissues derived from pharyngeal endoderm, FoxN1 expression is signature for the thymic epithelial lineage. Another is that expression of tissue-restricted Ag (TRA) is a unique feature of thymic epithelium. In this murine study, we evaluate the thymic expression of a subset of TRA, parathyroid hormone, calcitonin, and thyroglobulin, as part of an effort to better define the heterogeneity of medullary thymic epithelial cells. In this study, we demonstrate that both conventional and cystic epithelial cells display a history of FoxN1 expression using a cre-lox approach. We also document that extrathymic epithelial tissues that originate from pharyngeal endoderm also have a history of FoxN1 expression, indicating that FoxN1 expression per se is not a signature for the thymic lineage and suggesting that FoxN1 expression, whereas necessary for thymic epithelium, development, is not sufficient for this process to occur. Both cystic and conventional medullary thymic epithelial cells express these TRAs, as do extrathymic epithelial tissues that are not usually considered to be sources of these molecules. This finding supports the proposition that promiscuous gene expression is not unique to the thymus. Furthermore, the pattern of promiscuous gene expression in these extrathymic epithelia is consistent with developmental regulation processes and suggests that it is premature to discard the possibility that some promiscuous gene expression in the thymus reflects normal differentiation programs of epithelia.

Differentiation of regulatory Foxp3+ T cells in the thymic cortex.

Regulatory Foxp3(+) T cells (T(R)) are indispensable for preventing autoimmune pathology in multiple organs and tissues. During thymic differentiation T cell receptor (TCR)-ligand interactions within a certain increased affinity range, in conjunction with gammac-containing cytokine receptor signals, induce Foxp3 expression and thereby commit developing thymocytes to the T(R) lineage. The contribution of distinct MHC class II-expressing accessory cell types to the differentiation process of Foxp3(+) thymocytes remains controversial, because a unique role in this process has been ascribed to either thymic dendritic cells (tDC) or to medullary thymic epithelial cells (mTEC). Furthermore, it was suggested that the thymic medulla, where the bulk of the negative selection of self-reactive thymocytes takes place, provides a specialized microenvironment supporting T(R) differentiation. Here, we report that the cortex, as defined by cortical thymic epithelial cells (cTEC), is sufficient for supporting T(R) differentiation. MHC class II expression restricted to both cTEC and mTEC or to cTEC alone did not significantly affect the numbers of Foxp3(+) thymocytes. Furthermore, genetic or pharmacologic blockade of thymocyte migration resulted in a prominent accumulation of Foxp3(+) thymocytes in the cortex, demonstrating that secondary signals required for Foxp3 up-regulation exist in the cortex. Our results suggest that mTEC or tDC do not serve as a cell type singularly responsible for T(R) differentiation and that neither the cortex nor the medulla exclusively provides an environment suitable for Foxp3 induction. Instead, multiple accessory cell types probably contribute to the thymic generation of regulatory Foxp3(+) T cells.

Alterations of the medullary epithelial compartment in the Aire-deficient thymus: implications for programs of thymic epithelial differentiation.

A widely held model of thymic epithelial differentiation is based on patterns of keratin expression, where a K8(+)K5(+) progenitor gives rise to K8(+)K5/K14(-) cortical thymic epithelium (CTEC), and medullary thymic epithelium (MTEC) are K8(-)K5(+)K14(+). The thymic phenotype of p63-deficient mice indicates that p63 is an important regulator of proximal stages of thymic epithelial differentiation. In this study, we have examined several features of the thymic medullary compartment in wild-type and Aire-deficient thymi in an effort to integrate the proapoptotic activity of Aire with these different perspectives of TE differentiation. Patterns of keratin and p63 expression by MTEC described here are difficult to reconcile with postmitotic MTEC that express a K8(-)K14(+) phenotype and suggest that the patterns of p63 and keratin expression reflecting differentiation programs of other epithelial tissues provide a useful framework for revising models of TE differentiation. Alterations of the Aire(-/-) MTEC compartment included reduced expression of p63, increased frequency of MTEC expressing truncated Aire protein, and shifts in the pattern of keratin expression and epithelial morphology. These data suggest a scenario where cellular targets of Aire-mediated apoptosis are postmitotic MTEC that have not yet completed their terminal differentiation program. According to this view, the minor population of globular K8(+)K14(-/low) MTEC observed in the Aire(+/+) thymus and significantly expanded in the Aire(-/-) thymic medulla represent end-stage, terminally differentiated MTEC. These Aire-dependent alterations of the MTEC compartment suggest that the activity of Aire is not neutral with respect to the program of MTEC differentiation.

Evolutionary convergence in experimental Pseudomonas populations.

Model microbial systems provide opportunity to understand the genetic bases of ecological traits, their evolution, regulation and fitness contributions. Experimental populations of Pseudomonas fluorescens rapidly diverge in spatially structured microcosms producing a range of surface-colonising forms. Despite divergent molecular routes, wrinkly spreader (WS) niche specialist types overproduce a cellulosic polymer allowing mat formation at the air-liquid interface and access to oxygen. Given the range of ways by which cells can form mats, such phenotypic parallelism is unexpected. We deleted the cellulose-encoding genes from the ancestral genotype and asked whether this mutant could converge on an alternate phenotypic solution. Two new traits were discovered. The first involved an exopolysaccharide encoded by pgaABCD that functions as cell-cell glue similar to cellulose. The second involved an activator of an amidase (nlpD) that when defective causes cell chaining. Both types form mats, but were less fit in competition with cellulose-based WS types. Surprisingly, diguanylate cyclases linked to cellulose overexpression underpinned evolution of poly-beta-1,6-N-acetyl-d-glucosamine (PGA)-based mats. This prompted genetic analyses of the relationships between the diguanylate cyclases WspR, AwsR and MwsR, and both cellulose and PGA. Our results suggest that c-di-GMP regulatory networks may have been shaped by evolution to accommodate loss and gain of exopolysaccharide modules facilitating adaptation to new environments.

Adaptive evolution by spontaneous domain fusion and protein relocalization.

Knowledge of adaptive processes encompasses understanding the emergence of new genes. Computational analyses of genomes suggest that new genes can arise by domain swapping; however, empirical evidence has been lacking. Here we describe a set of nine independent deletion mutations that arose during selection experiments with the bacterium Pseudomonas fluorescens in which the membrane-spanning domain of a fatty acid desaturase became translationally fused to a cytosolic di-guanylate cyclase, generating an adaptive 'wrinkly spreader' phenotype. Detailed genetic analysis of one gene fusion shows that the mutant phenotype is caused by relocalization of the di-guanylate cyclase domain to the cell membrane. The relative ease by which this new gene arose, along with its functional and regulatory effects, provides a glimpse of mutational events and their consequences that are likely to have a role in the evolution of new genes.