William B. Ludington

Isolation and Characterization of a Protochordate Histocompatibility Locus

Histocompatibility—the ability of an organism to distinguish its own cells and tissue from those of another—is a universal phenomenon in the Metazoa. In vertebrates, histocompatibility is a function of the immune system controlled by a highly polymorphic major histocompatibility complex (MHC), which encodes proteins that target foreign molecules for immune cell recognition. The association of the MHC and immune function suggests an evolutionary relationship between metazoan histocompatibility and the origins of vertebrate immunity. However, the MHC of vertebrates is the only functionally characterized histocompatibility system; the mechanisms underlying this process in non-vertebrates are unknown. A primitive chordate, the ascidian Botryllus schlosseri, also undergoes a histocompatibility reaction controlled by a highly polymorphic locus. Here we describe the isolation of a candidate gene encoding an immunoglobulin superfamily member that, by itself, predicts the outcome of histocompatibility reactions. This is the first non-vertebrate histocompatibility gene described, and may provide insights into the evolution of vertebrate adaptive immunity.

Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model

Chlamydomonas reinhardtii IFT particle trains, important for flagella maintenance and assembly, are observed to decrease in size as a function of cilia length.

Organelle Size Equalization by a Constitutive Process

How cells control organelle size is an elusive problem. Two predominant models for size control can be distinguished: (1) induced control, where organelle genesis, maintenance, and disassembly are three separate programs that are activated in response to size change, and (2) constitutive control, where stable size results from the balance between continuous organelle assembly and disassembly. The problem has been studied in Chlamydomonas reinhardtii because the flagella are easy to measure, their size changes only in the length dimension, and the genetics are comparable to yeast. Length dynamics in Chlamydomonas flagella are quite robust: they maintain a length of about 12 μm and recover from amputation in about 90 min with a growth rate that decreases smoothly to zero as the length approaches 12 μm. Despite a wealth of experimental studies, existing data are consistent with both induced and constitutive control models for flagella. Here we developed novel microfluidic trapping and laser microsurgery techniques in Chlamydomonas to distinguish between length control models by measuring the two flagella on a single cell as they equilibrate after amputation of a single flagellum. The results suggest that cells equalize flagellar length by constitutive control.

Probabilistic Invasion Underlies Natural Gut Microbiome Stability

Species compositions of gut microbiomes impact host health [1-3], but the processes determining these compositions are largely unknown. An unexplained observation is that gut species composition varies widely between individuals but is largely stable over time within individuals [4, 5]. Stochastic factors during establishment may drive these alternative stable states (colonized versus non-colonized) [6, 7], which can influence susceptibility to pathogens, such as Clostridium difficile. Here we sought to quantify and model the dose response, dynamics, and stability of bacterial colonization in the fruit fly (Drosophila melanogaster) gut. Our precise, high-throughput technique revealed stable between-host variation in colonization when individual germ-free flies were fed their own natural commensals (including the probiotic Lactobacillus plantarum). Some flies were colonized while others remained germ-free even at extremely high bacterial doses. Thus, alternative stable states of colonization exist even in this low-complexity model of host-microbe interactions. These alternative states are driven by a fundamental asymmetry between the inoculum population and the stably colonized population that is mediated by spatial localization and a population bottleneck, which makes stochastic effects important by lowering the effective population size. Prior colonization with other bacteria reduced the chances of subsequent colonization, thus increasing the stability of higher-diversity guts. Therefore, stable gut diversity may be driven by inherently stochastic processes, which has important implications for combatting infectious diseases and for stably establishing probiotics in the gut.

A Systematic Comparison of Mathematical Models for Inherent Measurement of Ciliary Length: How a Cell Can Measure Length and Volume

Cells control organelle size with great precision and accuracy to maintain optimal physiology, but the mechanisms by which they do so are largely unknown. Cilia and flagella are simple organelles in which a single measurement, length, can represent size. Maintenance of flagellar length requires an active transport process known as intraflagellar transport, and previous measurements suggest that a length-dependent feedback regulates intraflagellar transport. But the question remains: how is a length-dependent signal produced to regulate intraflagellar transport appropriately? Several conceptual models have been suggested, but testing these models quantitatively requires that they be cast in mathematical form. Here, we derive a set of mathematical models that represent the main broad classes of hypothetical size-control mechanisms currently under consideration. We use these models to predict the relation between length and intraflagellar transport, and then compare the predicted relations for each model with experimental data. We find that three models-an initial bolus formation model, an ion current model, and a diffusion-based model-show particularly good agreement with available experimental data. The initial bolus and ion current models give mathematically equivalent predictions for length control, but fluorescence recovery after photobleaching experiments rule out the initial bolus model, suggesting that either the ion current model or a diffusion-based model is more likely correct. The general biophysical principles of the ion current and diffusion-based models presented here to measure cilia and flagellar length can be generalized to measure any membrane-bound organelle volume, such as the nucleus and endoplasmic reticulum.

Stable Host Gene Expression in the Gut of Adult Drosophila melanogaster with Different Bacterial Mono-Associations

There is growing evidence that the microbes found in the digestive tracts of animals influence host biology, but we still do not understand how they accomplish this. Here, we evaluated how different microbial species commonly associated with laboratory-reared Drosophila melanogaster impact host biology at the level of gene expression in the dissected adult gut and in the entire adult organism. We observed that guts from animals associated from the embryonic stage with either zero, one or three bacterial species demonstrated indistinguishable transcriptional profiles. Additionally, we found that the gut transcriptional profiles of animals reared in the presence of the yeast Saccharomyces cerevisiae alone or in combination with bacteria could recapitulate those of conventionally-reared animals. In contrast, we found whole body transcriptional profiles of conventionally-reared animals were distinct from all of the treatments tested. Our data suggest that adult flies are insensitive to the ingestion of the bacteria found in their gut, but that prior to adulthood, different microbes impact the host in ways that lead to global transcriptional differences observable across the whole adult body.

Automated analysis of intracellular motion using kymographs in 1, 2, and 3 dimensions

In this paper we use kymographs and computational image processing to convert 3-D video microscopy data of intracellular motion into 1-D time series data for further analysis. Because standard tools exist for time series analysis, this method allows us to produce robust quantitative results from otherwise visual data. The kymograph-based approach has an additional advantage over standard particle-tracking and flow-based image quantification algorithms in that we can average out camera noise over the spatial axis of the kymograph. The method has the disadvantage that it removes all spatial information. For this reason we see this method as a complement to rather than a replacement of standard tracking algorithms. The standard problem we are trying to address in our work is how fluorescent proteins in one cellular compartment are injected into another cellular compartment. The proteins travel at constant speed along a fixed spatial path, so a 2-D kymograph produced from a trace along this fixed path will tell us about the injection history into this second compartment. Our algorithm works by first taking a Radon transform of the input 2-D kymograph. We next make synthetic kymographs by backprojection. The angle with the best correlation between the original kymograph and the backprojection determines the dominant speed of the moving particles as well as the angle of the 1-D projected time series. Time series are then analyzed with standard tools to determine the peak size distribution, the peak interval distribution, the autocorrelation and the power spectrum.

High-dimensional microbiome interactions shape host fitness

Gut bacteria can affect key aspects of host fitness, such as development, fecundity, and lifespan, while the host in turn shapes the gut microbiome. Microbiomes co-evolve with their hosts and have been implicated in host speciation. However, it is unclear to what extent individual species versus community interactions within the microbiome are linked to host fitness. Here we combinatorially dissect the natural microbiome of Drosophila melanogaster and reveal that interactions between bacteria shape host fitness through life history tradeoffs. We find that the same microbial interactions that shape host fitness also shape microbiome abundances, suggesting a potential evolutionary mechanism by which microbiome communities (rather than just individual species) may be intertwined in co-selection with their hosts. Empirically, we made germ-free flies colonized with each possible combination of the five core species of fly gut bacteria. We measured the resulting bacterial community abundances and fly fitness traits including development, reproduction, and lifespan. The fly gut promoted bacterial diversity, which in turn accelerated development, reproduction, and aging: flies that reproduced more died sooner. From these measurements we calculated the impact of bacterial interactions on fly fitness by adapting the mathematics of genetic epistasis to the microbiome. Host physiology phenotypes were highly dependent on interactions between bacterial species. Higher-order interactions (involving 3, 4, and 5 species) were widely prevalent and impacted both host physiology and the maintenance of gut diversity. The parallel impacts of bacterial interactions on the microbiome and on host fitness suggest that microbiome interactions may be key drivers of evolution. Significance All multicellular organisms have associated microbial communities called microbiomes that can influence the physiology and fitness of their host. It is unclear to what extent individual microbial species versus ecology of the microbiome influences fitness of the host. Here we mapped all the possible interactions between individual species of bacteria with each other and with the host’s physiology. Our approach revealed that the same bacterial interactions that shape microbiome abundances also shape host fitness traits. This relationship provides a feedback that may favor the emergence of co-evolving microbiome-host units.

Assessing biosynthetic potential of agricultural groundwater through metagenomic sequencing: A diverse anammox community dominates nitrate-rich groundwater

Background Climate change produces extremes in both temperature and precipitation causing increased drought severity and increased reliance on groundwater resources. Agricultural practices, which rely on groundwater, are sensitive to but also sources of contaminants, including nitrate. How agricultural contamination drives groundwater geochemistry through microbial metabolism is poorly understood. Methods On an active cow dairy in the Central Valley of California, we sampled groundwater from three wells at depths of 4.3 m (two wells) and 100 m (one well) below ground surface (bgs) as well as an effluent surface water lagoon that fertilizes surrounding corn fields. We analyzed the samples for concentrations of solutes, heavy metals, and USDA pathogenic bacteria of the Escherichia coli and Enterococcus groups as part of a long term groundwater monitoring study. Whole metagenome shotgun sequencing and assembly revealed taxonomic composition and metabolic potential of the community. Results Elevated nitrate and dissolved organic carbon occurred at 4.3m but not at 100m bgs. Metagenomics confirmed chemical observations and revealed several Planctomycete genomes, including a new Brocadiaceae lineage and a likely Planctomycetes OM190, as well novel diversity and high abundance of nano-prokaryotes from the Candidate Phyla Radiation (CPR), the Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea (DPANN) and the Thaumarchaeota, Aigarchaeota, Crenarchaeota, Korarchaeota (TACK) superphyla. Pathway analysis suggests community interactions based on complimentary primary metabolic pathways and abundant secondary metabolite operons encoding antimicrobials and quorum sensing systems. Conclusions The metagenomes show strong resemblance to activated sludge communities from a nitrogen removal reactor at a wastewater treatment plant, suggesting that natural bioremediation occurs through microbial metabolism. Elevated nitrate and rich secondary metabolite biosynthetic capacity suggest incomplete remediation and the potential for novel pharmacologically active compounds.

Diet influences host–microbiota associations in Drosophila

Gut microbes were previously suggested to influence mate preference in Drosophila melanogaster (1). Mate selectivity depended on the microbiota associated with flies after prior generations were maintained on different diets \[cornmeal–molasses–yeast (CMY) versus starch\] (1). Subsequent studies attempted to repeat these findings with contrasting success (2, 3). We suggest that a nonstandardized, transient microbiota—and how it is influenced by diet and dietary additives—might account for the conflicting results. A point of contention between the studies (1⇓–3) is that a fungicide used in fly media, methylparaben (mp)—also known as Tegosept or Nipagin M—might affect bacterial growth and thus microbiota composition (4, 5). Although Leftwich et al. (4) argued that moderate mp (up to 0.3%) should not alter microbiota, a previous study suggested that high mp levels (∼0.5%) can impact microbiota diversity (6). How mp concentration affects individual, fly-associated microbes on fly media has not been systematically addressed. We found that using mp over 0.1% in fly medium severely restricts growth of some yeast and Acetobacter … [↵][1]3To whom correspondence may be addressed. Email: wja{at}scripps.edu or will.ludington{at}berkeley.edu. [1]: #xref-corresp-1-1