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The central hypothesis of the genotype–phenotype relationship is that the phenotype of a developing organism (i.e., its set of observable attributes) depends on its genome and the environment. However, as we learn more about the genetics and biochemistry of living systems, our understanding does not fully extend to the complex multiscale nature of how cells move, interact, and organize; this gap in understanding is referred to as the genotype-to-phenotype problem. The physics of soft matter sets the background on which living organisms evolved, and the cell environment is a strong determinant of cell phenotype. This inevitably leads to challenges as the full function of many genes, and the diversity of cellular behaviors cannot be assessed without wide screens of environmental conditions. Cellular mechanobiology is an emerging field that provides methodologies to understand how cells integrate chemical and physical environmental stress and signals, and how they are transduced to control cell function. Biofilm forming bacteria represent an attractive model because they are fast growing, genetically malleable and can display sophisticated self-organizing developmental behaviors similar to those found in higher organisms. Here, we propose mechanobiology as a new area of study in prokaryotic systems and describe its potential for unveiling new links between an organism's genome and phenome. 

ABSTRACT Myxococcus xanthus copes with starvation by producing fruiting bodies filled with dormant and stress-resistant spores. Here, we aimed to better define the gene regulatory network associated with Nla28, a transcriptional activator/enhancer binding protein (EBP) and a key regulator of the early starvation response. Previous work showed that Nla28 directly regulates EBP genes that are important for fruiting body development. However, the Nla28 regulatory network is likely to be much larger because hundreds of starvation-induced genes are downregulated in a nla28 mutant strain. To identify candidates for direct Nla28-mediated transcription, we analyzed the downregulated genes using a bioinformatics approach. Nine potential Nla28 target promoters (29 genes) were discovered. The results of in vitro promoter binding assays, coupled with in vitro and in vivo mutational analyses, suggested that the nine promoters along with three previously identified EBP gene promoters were indeed in vivo targets of Nla28. These results also suggested that Nla28 used tandem, imperfect repeats of an 8-bp sequence for promoter binding. Interestingly, eight of the new Nla28 target promoters were predicted to be intragenic. Based on mutational analyses, the newly identified Nla28 target loci contained at least one gene that was important for starvation-induced development. Most of these loci contained genes predicted to be involved in metabolic or defense-related functions. Using the consensus Nla28 binding sequence, bioinformatics, and expression profiling, 58 additional promoters and 102 genes were tagged as potential Nla28 targets. Among these putative Nla28 targets, functions, such as regulatory, metabolic, and cell envelope biogenesis, were assigned to many genes. IMPORTANCE In bacteria, starvation leads to profound changes in behavior and physiology. Some of these changes have economic and health implications because the starvation response has been linked to the formation of biofilms, virulence, and antibiotic resistance. To better understand how starvation contributes to changes in bacterial physiology and resistance, we identified the putative starvation-induced gene regulatory network associated with Nla28, a transcriptional activator from the bacterium Myxoccocus xanthus . We determined the mechanism by which starvation-responsive genes were activated by Nla28 and showed that several of the genes were important for the formation of a highly resistant cell type. 

A source generates time-stamped update packets that are sent to a server and then forwarded to a monitor. This occurs in the presence of an adversary that can infer information about the source by observing the output process of the server. The server wishes to release updates in a timely way to the monitor but also wishes to minimize the information leaked to the adversary. We analyze the trade-off between the age of information (AoI) and the maximal leakage for systems in which the source generates updates as a Bernoulli process. For a time slotted system in which sending an update requires one slot, we consider three server policies: (1) Memoryless with Bernoulli Thinning (MBT): arriving updates are queued with some probability and head-of-line update is released after a geometric holding time; (2) Deterministic Accumulate-and-Dump (DAD): the most recently generated update (if any) is released after a fixed time; (3) Random Accumulate-and-Dump (RAD): the most recently generated update (if any) is released after a geometric waiting time. We show that for the same maximal leakage rate, the DAD policy achieves lower age compared to the other two policies but is restricted to discrete age-leakage operating points. 

To better understand tick ecology in Virginia and the increasing Lyme disease incidence in western Virginia, a comparative phenological study was conducted in which monthly collections were performed at twelve sampling locations in southwestern Virginia (high Lyme disease incidence) and 18 equivalent sampling locations in southeastern Virginia (low Lyme disease incidence) for one year. In western Virginia, we also explored the effect of elevation on collection rates of Ixodes scapularis Say (Acari: Ixodidae) and Amblyomma americanum (L.) (Acari: Ixodidae). In total, 35,438 ticks were collected (33,106 A. americanum; 2,052 I. scapularis; 134 Ixodes affinis Neumann [Acari: Ixodidae]; 84 Dermacentor variabilis [Say] [Acari: Ixodidae]; 49 Dermacentor albipictus [Packard] [Acari: Ixodidae]; 10 Haemaphysalis leporispalustris [Packard] [Acari: Ixodidae]; 2 Ixodes brunneus Koch [Acari: Ixodidae]; 1 Haemaphysalis longicornis Neumann [Acari: Ixodidae]). Within southwestern Virginia, Ixodes scapularis collection rates were not influenced by elevation, unlike A. americanum which were collected more frequently at lower elevations (e.g., below 500 m). Notably, I. scapularis larvae and nymphs were commonly collected in southwestern Virginia (indicating that they were questing on or above the leaf litter) but not in southeastern Virginia. Questing on or above the leaf litter is primarily associated with northern populations of I. scapularis. These findings may support the hypothesis that I. scapularis from the northeastern United States are migrating into western Virginia and contributing to the higher incidence of Lyme disease in this region. This comparative phenological study underscores the value of these types of studies and the need for additional research to further understand the rapidly changing tick-borne disease dynamics in Virginia.

 

Abstract Molecular interactions are studied as independent networks in systems biology. However, molecular networks do not exist independently of each other. In a network of networks approach (called multiplex), we study the joint organization of transcriptional regulatory network (TRN) and protein–protein interaction (PPI) network. We find that TRN and PPI are non-randomly coupled across five different eukaryotic species. Gene degrees in TRN (number of downstream genes) are positively correlated with protein degrees in PPI (number of interacting protein partners). Gene–gene and protein–protein interactions in TRN and PPI, respectively, also non-randomly overlap. These design principles are conserved across the five eukaryotic species. Robustness of the TRN–PPI multiplex is dependent on this coupling. Functionally important genes and proteins, such as essential, disease-related and those interacting with pathogen proteins, are preferentially situated in important parts of the human multiplex with highly overlapping interactions. We unveil the multiplex architecture of TRN and PPI. Multiplex architecture may thus define a general framework for studying molecular networks. This approach may uncover the building blocks of the hierarchical organization of molecular interactions. 

A source node forwards fresh status updates as a point process to a network of observer nodes. Within the network of observers, these updates are forwarded as point processes from node to node. Each node wishes its knowledge of the source to be as timely as possible. In this network, timeliness at each node is measured by an age of information metric: how old is the timestamp of the freshest received update. This work extends a method for evaluating the average age at each node in the network when nodes forward updates using a memoryless gossip protocol. This method is then demonstrated by age analysis for a simple network. 

A source node updates its status as a point process and also forwards its updates to a network of observer nodes. Within the network of observers, these updates are forwarded as point processes from node to node. Each node wishes its knowledge of the source to be as timely as possible. In this network, timeliness is measured by a discrete form of age of information: each status change at the source is referred to as a version and the age at a node is how many versions out of date is its most recent update from the source. This work introduces a method for evaluating the average version age at each node in the network when nodes forward updates using a memoryless gossip protocol. This method is then demonstrated by version age analysis for a collection of simple networks. For gossip on a complete graph with symmetric updating rates, it is shown that each node has average age that grows as the logarithm of the network size. 

ABSTRACT Myxococcus xanthus is a bacterium that lives on surfaces as a predatory biofilm called a swarm. As a growing swarm feeds on prey and expands, it displays dynamic multicellular patterns such as traveling waves called ripples and branching protrusions called flares. The rate at which a swarm expands across a surface, and the emergence of the coexisting patterns, are all controlled through coordinated cell movement. M. xanthus cells move using two motility systems known as adventurous (A) and social (S). Both are involved in swarm expansion and pattern formation. In this study, we describe a set of M. xanthus swarming genotype-to-phenotype associations that include both genetic and environmental perturbations. We identified new features of the swarming phenotype, recorded and measured swarm expansion using time-lapse microscopy, and compared the impact of mutations on different surfaces. These observations and analyses have increased our ability to discriminate between swarming phenotypes and provided context that allows us to identify some phenotypes as improbable outliers within the M. xanthus swarming phenome. IMPORTANCE Myxococcus xanthus grows on surfaces as a predatory biofilm called a swarm. In nature, a feeding swarm expands by moving over and consuming prey bacteria. In the laboratory, a swarm is created by spotting cell suspension onto nutrient agar in lieu of prey. The suspended cells quickly settle on the surface as the liquid is absorbed into the agar, and the new swarm then expands radially. An assay that measures the expansion rate of a swarm of mutant cells is the first, and sometimes only, measurement used to decide whether a particular mutation impacts swarm motility. We have broadened the scope of this assay by increasing the accuracy of measurements and introducing prey, resulting in new identifiable and quantifiable features that can be used to improve genotype-to-phenotype associations. 

The ability of bacteria to colonize and grow on different surfaces is an essential process for biofilm development. Here, we report the use of synthetic hydrogels with tunable stiffness and porosity to assess physical effects of the substrate on biofilm development. Using time-lapse microscopy to track the growth of expanding Serratia marcescens colonies, we find that biofilm colony growth can increase with increasing substrate stiffness, unlike what is found on traditional agar substrates. Using traction force microscopy-based techniques, we find that biofilms exert transient stresses correlated over length scales much larger than a single bacterium, and that the magnitude of these forces also increases with increasing substrate stiffness. Our results are consistent with a model of biofilm development in which the interplay between osmotic pressure arising from the biofilm and the poroelastic response of the underlying substrate controls biofilm growth and morphology.