Dispersal: a matter of scale.

Population density around the natal site is often invoked as an explanation for variation in dispersal distance, with the expectation that competition for limiting resources, coupled with increased intra-specific aggression at high densities, should drive changes in dispersal distances. However, tests of the density-dependent dispersal hypothesis in long-lived vertebrates have yielded mixed results. Furthermore, conclusions from dispersal studies may depend on the spatial and temporal scales at which density and dispersal patterns are examined, yet multi-scale studies of dispersal are rare. Here, we present the findings of a long-term study examining factors influencing natal dispersal distances for the non-migratory population of Peregrine Falcons (Falco peregrinus) in the British Isles across distinct spatial and temporal scales. Our smallest scale study included Peregrines ringed as nestlings and subsequently recaptured alive in south Scotland-north England, an area that was intensively studied during the time periods 1974-1982 and 2002-2016. Second, we examined dispersal patterns of birds ringed as nestlings in south Scotland-north England, but subsequently recaptured alive or recovered dead anywhere in the British Isles. Finally, we examined the natal dispersal patterns for Peregrines ringed and recaptured or recovered anywhere in the British Isles from 1964 to 2016. Consistent with prior findings, females dispersed farther than males across all scales. However, the patterns of dispersal were strongly scale dependent. Specifically, we found a lack of a discernible relationship between index of density and dispersal distance in the limited study area, but when region-wide recaptures and recoveries were included in the analyses, a negative relationship was revealed. Our results suggest that conclusions of dispersal studies may be scale dependent, highlighting the importance of spatial and temporal scales in examining and interpreting the relationship between population density and dispersal patterns.

Gut Microbiome of Coexisting BaAka Pygmies and Bantu Reflects Gradients of Traditional Subsistence Patterns.

To understand how the gut microbiome is impacted by human adaptation to varying environments, we explored gut bacterial communities in the BaAka rainforest hunter-gatherers and their agriculturalist Bantu neighbors in the Central African Republic. Although the microbiome of both groups is compositionally similar, hunter-gatherers harbor increased abundance of Prevotellaceae, Treponema, and Clostridiaceae, while the Bantu gut microbiome is dominated by Firmicutes. Comparisons with US Americans reveal microbiome differences between Africans and westerners but show western-like features in the Bantu, including an increased abundance of predictive carbohydrate and xenobiotic metabolic pathways. In contrast, the hunter-gatherer gut shows increased abundance of predicted virulence, amino acid, and vitamin metabolism functions, as well as dominance of lipid and amino-acid-derived metabolites, as determined through metabolomics. Our results demonstrate gradients of traditional subsistence patterns in two neighboring African groups and highlight the adaptability of the microbiome in response to host ecology.

Variation in Rural African Gut Microbiota Is Strongly Correlated with Colonization by Entamoeba and Subsistence.

The human gut microbiota is impacted by host nutrition and health status and therefore represents a potentially adaptive phenotype influenced by metabolic and immune constraints. Previous studies contrasting rural populations in developing countries to urban industrialized ones have shown that industrialization is strongly correlated with patterns in human gut microbiota; however, we know little about the relative contribution of factors such as climate, diet, medicine, hygiene practices, host genetics, and parasitism. Here, we focus on fine-scale comparisons of African rural populations in order to (i) contrast the gut microbiota of populations inhabiting similar environments but having different traditional subsistence modes and either shared or distinct genetic ancestry, and (ii) examine the relationship between gut parasites and bacterial communities. Characterizing the fecal microbiota of Pygmy hunter-gatherers as well as Bantu individuals from both farming and fishing populations in Southwest Cameroon, we found that the gut parasite Entamoeba is significantly correlated with microbiome composition and diversity. We show that across populations, colonization by this protozoa can be predicted with 79% accuracy based on the composition of an individual's gut microbiota, and that several of the taxa most important for distinguishing Entamoeba absence or presence are signature taxa for autoimmune disorders. We also found gut communities to vary significantly with subsistence mode, notably with some taxa previously shown to be enriched in other hunter-gatherers groups (in Tanzania and Peru) also discriminating hunter-gatherers from neighboring farming or fishing populations in Cameroon.

Ecological dynamics and complex interactions of Agrobacterium megaplasmids.

As with many pathogenic bacteria, agrobacterial plant pathogens carry most of their virulence functions on a horizontally transmissible genetic element. The tumor-inducing (Ti) plasmid encodes the majority of virulence functions for the crown gall agent Agrobacterium tumefaciens. This includes the vir genes which drive genetic transformation of host cells and the catabolic genes needed to utilize the opines produced by infected plants. The Ti plasmid also encodes, an opine-dependent quorum sensing system that tightly regulates Ti plasmid copy number and its conjugal transfer to other agrobacteria. Many natural agrobacteria are avirulent, lacking the Ti plasmid. The burden of harboring the Ti plasmid depends on the environmental context. Away from diseased hosts, plasmid costs are low but the benefit of the plasmid is also absent. Consequently, plasmidless genotypes are favored. On infected plants the costs of the Ti plasmid can be very high, but balanced by the opine benefits, locally favoring plasmid bearing cells. Cheating derivatives which do not incur virulence costs but can benefit from opines are favored on infected plants and in most other environments, and these are frequently isolated from nature. Many agrobacteria also harbor an At plasmid which can stably coexist with a Ti plasmid. At plasmid genes are less well characterized but in general facilitate metabolic activities in the rhizosphere and bulk soil, such as the ability to breakdown plant exudates. Examination of A. tumefaciens C58, revealed that harboring its At plasmid is much more costly than harboring it's Ti plasmid, but conversely the At plasmid is extremely difficult to cure. The interactions between these co-resident plasmids are complex, and depend on environmental context. However, the presence of a Ti plasmid appears to mitigate At plasmid costs, consistent with the high frequency with which they are found together.

Agrobacterium tumefaciens exoR controls acid response genes and impacts exopolysaccharide synthesis, horizontal gene transfer, and virulence gene expression.

Agrobacterium tumefaciens is a facultative plant pathogen and the causative agent of crown gall disease. The initial stage of infection involves attachment to plant tissues, and subsequently, biofilms may form at these sites. This study focuses on the periplasmic ExoR regulator, which was identified based on the severe biofilm deficiency of A. tumefaciens exoR mutants. Genome-wide expression analysis was performed to elucidate the complete ExoR regulon. Overproduction of the exopolysaccharide succinoglycan is a dramatic phenotype of exoR mutants. Comparative expression analyses revealed that the core ExoR regulon is unaffected by succinoglycan synthesis. Several findings are consistent with previous observations: genes involved in succinoglycan biosynthesis, motility, and type VI secretion are differentially expressed in the ΔexoR mutant. In addition, these studies revealed new functional categories regulated by ExoR, including genes related to virulence, conjugation of the pAtC58 megaplasmid, ABC transporters, and cell envelope architecture. To address how ExoR exerts a broad impact on gene expression from its periplasmic location, a genetic screen was performed to isolate suppressor mutants that mitigate the exoR motility phenotype and identify downstream components of the ExoR regulatory pathway. This suppression analysis identified the acid-sensing two-component system ChvG-ChvI, and the suppressor mutant phenotypes suggest that all or most of the characteristic exoR properties are mediated through ChvG-ChvI. Subsequent analysis indicates that exoR mutants are simulating a response to acidic conditions, even in neutral media. This work expands the model for ExoR regulation in A. tumefaciens and underscores the global role that this regulator plays on gene expression.

Non-additive costs and interactions alter the competitive dynamics of co-occurring ecologically distinct plasmids.

Plasmids play an important role in shaping bacterial evolution and adaptation to heterogeneous environments. As modular genetic elements that are often conjugative, the selective pressures that act on plasmid-borne genes are distinct from those that act on the chromosome. Many bacteria are co-infected by multiple plasmids that impart niche-specific phenotypes. Thus, in addition to host-plasmid dynamics, interactions between co-infecting plasmids are likely to be important drivers of plasmid population dynamics, evolution and ecology. Agrobacterium tumefaciens is a facultative plant pathogen that commonly harbours two distinct megaplasmids. Virulence depends on the presence of the tumour-inducing (Ti) plasmid, with benefits that are primarily restricted to the disease environment. Here, we demonstrate that a second megaplasmid, the At plasmid, confers a competitive advantage in the rhizosphere. To assess the individual and interactive costs of these plasmids, we generated four isogenic derivatives: plasmidless, pAt only, pTi only and pAtpTi, and performed pairwise competitions under carbon-limiting conditions. These studies reveal a low cost to the virulence plasmid when outside of the disease environment, and a strikingly high cost to the At plasmid. In addition, the costs of pAt and pTi in the same host were significantly lower than predicted based on single plasmid costs, signifying the first demonstration of non-additivity between naturally occurring co-resident plasmids. Based on these empirically demonstrated costs and benefits, we developed a resource-consumer model to generate predictions about the frequencies of these genotypes in relevant environments, showing that non-additivity between co-residing plasmids allows for their stable coexistence across environments.

Large deletions in the pAtC58 megaplasmid of Agrobacterium tumefaciens can confer reduced carriage cost and increased expression of virulence genes.

The accessory plasmid pAtC58 of the common laboratory strain of Agrobacterium tumefaciens confers numerous catabolic functions and has been proposed to play a role in virulence. Genomic sequencing of evolved laboratory strains of A. tumefaciens revealed the presence of multiple deletion events in the At plasmid, with reductions in plasmid size ranging from 25% to 30% (115-194 kb). Flanking both ends of the sites of these deletions is a short-nucleotide repeat sequence that is in a single copy in the deleted plasmids, characteristic of a phage- or transposon-mediated deletion event. This repeat sequence is widespread throughout the C58 genome, but concentrated on the At plasmid, suggesting its frequency to be nonrandom. In this study, we assess the prevalence of the larger of these deletions in multiple C58 derivatives and characterize its functional significance. We find that in addition to elevating virulence gene expression, this deletion is associated with a significantly reduced carriage cost to the cell. These observations are a clear demonstration of the dynamic nature of the bacterial genome and suggest a mechanism for genetic plasticity of these costly but otherwise stable plasmids. Additionally, this phenomenon could be the basis for some of the dramatic recombination events so ubiquitous within and among megaplasmids.

Isolation and cultivation of Agrobacterium species from natural sources.

Selected species of the genus Agrobacterium have been extensively studied in the laboratory, but far less is known regarding their natural distribution. Agrobacterium can be isolated from a variety of environments, but identifying the bacteria is challenging and involves physiological assays as well as molecular diagnostics.

Microbial population and community dynamics on plant roots and their feedbacks on plant communities.

The composition of the soil microbial community can be altered dramatically due to association with individual plant species, and these effects on the microbial community can have important feedbacks on plant ecology. Negative plant-soil feedback plays primary roles in maintaining plant community diversity, whereas positive plant-soil feedback may cause community conversion. Host-specific differentiation of the microbial community results from the trade-offs associated with overcoming plant defense and the specific benefits associated with plant rewards. Accumulation of host-specific pathogens likely generates negative feedback on the plant, while changes in the density of microbial mutualists likely generate positive feedback. However, the competitive dynamics among microbes depends on the multidimensional costs of virulence and mutualism, the fine-scale spatial structure within plant roots, and active plant allocation and localized defense. Because of this, incorporating a full view of microbial dynamics is essential to explaining the dynamics of plant-soil feedbacks and therefore plant community ecology.

Genetic manipulation of Agrobacterium.

Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens, this is called the Ti (tumor-inducing) plasmid. During pathogenesis virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome, and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool and is also a model microorganism that has been well studied for host-microbe associations, horizontal gene transfer, cell-cell communication, and biofilm formation. This unit describes standard protocols for genetic manipulation of A. tumefaciens.

Phenotypic analyses of Agrobacterium.

Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens this is called the Ti (tumor-inducing) plasmid. During pathogenesis virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome, and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool and is also a model microorganism that has been well studied for host-microbe associations, horizontal gene transfer, cell-cell communication, and biofilm formation. This unit describes standard protocols for simple phenotypic characterizations of A. tumefaciens.

Laboratory maintenance of Agrobacterium.

Agrobacterium species are plant-associated relatives of the rhizobia. Several species cause plant diseases such as crown gall and hairy root, although there are also avirulent species. A. tumefaciens is the most intensively studied species and causes crown gall, a neoplastic disease that occurs on a variety of plants. Virulence is specified by large plasmids, and in the case of A. tumefaciens this is called the Ti (tumor-inducing) plasmid. During pathogenesis, virulent agrobacteria copy a segment of the Ti plasmid and transfer it to the plant, where it subsequently integrates into the plant genome and expresses genes that result in the disease symptoms. A. tumefaciens has been used extensively as a plant genetic engineering tool, and is also a model microorganism that has been well studied for host-microbe associations, horizontal gene transfer, cell-cell communication, and biofilm formation. This unit describes standard protocols for laboratory cultivation of A. tumefaciens.

Herpes simplex virus blocks Fas-mediated apoptosis independent of viral activation of NF-kappaB in human epithelial HEp-2 cells.

The goal of our study was to characterize the apoptotic response of herpes simplex virus (HSV)-infected, human epithelial HEp-2 cells to extrinsic treatments through the Fas receptor. Initially, we defined the Fas response of these cells. We found the following: (1) Treatment of HEp-2 cells with anti-Fas antibody or Fas ligand (FasL) alone did not induce apoptosis. (2) In addition, these inducers did not activate NF-kappaB in these cells. (3) The addition of cycloheximide (CHX) during these treatments caused a dramatic increase in programmed cell death. (4) HEp-2 cells infected with HSV for 6 h prior to anti-Fas plus CHX treatment were nonapoptotic, and (5) these cells possessed nuclear NFkappaB. (6) HSV blocked anti-Fas or FasL plus CHX-induced apoptosis in HEp-2 cells that stably expressed a dominant-negative form of IkappaBalpha. These results indicate that HSV infection can block the process of Fas-mediated apoptosis through a mechanism that is independent of viral activation of NFkappaB. Our findings help define the molecular mechanisms involved in HSV evasion of the cytokine-driven, innate immune response in human epithelial cells.

Herpes simplex virus: propagation, quantification, and storage.

Herpes simplex virus (HSV) is the prototype of a family of large, enveloped, double-stranded DNA viruses, the Herpesviridae, which cause significant morbidity and mortality in humans. Productive replication of HSV in cells in culture results in definitive changes in cellular physiology and metabolism, ultimately leading to lysis. These definitive aspects of viral-host interactions enable diagnosis of HSV infections. In this unit, a series of methods are described for the propagation, quantification, and storage of HSV. Infectious center assays are used to measure the titers of HSV stocks. In addition, immunological methods are described for documenting the accumulation of viral polypeptides in infected whole cell extracts, as well as in situ using indirect immunofluorescence. These techniques should be beneficial to basic research virologists utilizing standard laboratory HSV strains, as well as clinical microbiologists interested in characterizing HSV isolated from patients.

Herpes simplex virus infection and apoptosis.

Consequences of human herpes simplex virus (HSV) infection include the induction of apoptosis and the concomitant synthesis of proteins which act to block this process from killing the infected cell. Recent data has clarified our current understanding of the mechanisms of induction and prevention of apoptosis by HSV. These findings emphasize the fact that modulation of apoptosis by HSV during infection is a multicomponent phenomenon. We review recent evidence showing how this important human pathogen modulates the fundamental cell death process.