Spatial regulation is often encountered as a component of multi-tiered regulatory systems in eukaryotes, where processes are readily segregated by organelle boundaries. Well-characterized examples of spatial regulation are less common in bacteria. Low-fidelity DNA polymerase V (UmuD′2C) is produced in Escherichia coli as part of the bacterial SOS response to DNA damage. Due to the mutagenic potential of this enzyme, pol V activity is controlled by means of an elaborate regulatory system at transcriptional and posttranslational levels. Using single-molecule fluorescence microscopy to visualize UmuC inside living cells in space and time, we now show that pol V is also subject to a novel form of spatial regulation. After an initial delay (~ 45 min) post UV irradiation, UmuC is synthesized, but is not immediately activated. Instead, it is sequestered at the inner cell membrane. The release of UmuC into the cytosol requires the RecA* nucleoprotein filament-mediated cleavage of UmuD→UmuD′. Classic SOS damage response mutants either block [umuD(K97A)] or constitutively stimulate [recA(E38K)] UmuC release from the membrane. Foci of mutagenically active pol V Mut (UmuD′2C-RecA-ATP) formed in the cytosol after UV irradiation do not co-localize with pol III replisomes, suggesting a capacity to promote translesion DNA synthesis at lesions skipped over by DNA polymerase III. In effect, at least three molecular mechanisms limit the amount of time that pol V has to access DNA: (1) transcriptional and posttranslational regulation that initially keep the intracellular levels of pol V to a minimum; (2) spatial regulation via transient sequestration of UmuC at the membrane, which further delays pol V activation; and (3) the hydrolytic activity of a recently discovered pol V Mut ATPase function that limits active polymerase time on the chromosomal template.

Highlights d Quantum-dot technology can be used to track trading strategies of mycorrhizal fungi d Increasing exposure to inequality stimulates trade and resource movement d Fungal trade strategies are not uniform across the symbiotic network d Fungi can capitalize on trade by first moving resources to areas of high demand

Soil is likely the most complex ecosystem on earth. Despite the global importance and extraordinary diversity of soils, they have been notoriously challenging to study. We show how pioneering microfluidic techniques provide new ways of studying soil microbial ecology by allowing simulation and manipulation of chemical conditions and physical structures at the microscale in soil model habitats.

a b s t r a c tWe describe the purification, crystallization and structure for the GTP-binding domain of human septin 7 (SEPT7G). We show that it forms filaments within the crystal lattice which employ both the G and NC interfaces, similar to those seen in the hetero-filament of SEPT2/6/7. The NC interface is considered promiscuous as it is absent from the hetero-filament. Such promiscuity could provide the potential for permuting monomers along a filament in order to generate diversity in heteropolymers. On the other hand, our results suggest that the G and NC interfaces may be necessary but insufficient for determining correct hetero-filament assembly. Structured summary of protein interactions:SEPT7G and SEPT7G bind by X-ray crystallography (View interaction).

Recent technical advances have made it possible to visualize single molecules inside live cells. Microscopes with single-molecule sensitivity enable the imaging of low-abundance proteins, allowing for a quantitative characterization of molecular properties. Such data sets contain information on a wide spectrum of important molecular properties, with different aspects highlighted in different imaging strategies. The time-lapsed acquisition of images provides information on protein dynamics over long time scales, giving insight into expression dynamics and localization properties. Rapid burst imaging reveals properties of individual molecules in real-time, informing on their diffusion characteristics, binding dynamics and stoichiometries within complexes. This richness of information, however, adds significant complexity to analysis protocols. In general, large datasets of images must be collected and processed in order to produce statistically robust results and identify rare events. More importantly, as live-cell single-molecule measurements remain on the cutting edge of imaging, few protocols for analysis have been established and thus analysis strategies often need to be explored for each individual scenario. Existing analysis packages are geared towards either single-cell imaging data or in vitro single-molecule data and typically operate with highly specific algorithms developed for particular situations. Our tool, iSBatch, instead allows users to exploit the inherent flexibility of the popular open-source package ImageJ, providing a hierarchical framework in which existing plugins or custom macros may be executed over entire datasets or portions thereof. This strategy affords users freedom to explore new analysis protocols within large imaging datasets, while maintaining hierarchical relationships between experiments, samples, fields of view, cells, and individual molecules.

Tagging of individual proteins with genetically encoded fluorescent proteins (FPs) has been used extensively to study localization and interactions in live cells. Recent developments in single-molecule localization microscopy have enabled the dynamic visualization of individual tagged proteins inside living cells. However, tagging proteins with FPs is not without problems: formation of insoluble aggregates and inhibition of native functions of the protein are well-known issues. Previously reported artifacts manifest themselves at all expression levels of the FP-tagged proteins, making the design of control experiments relatively straightforward. Here, we describe a previously uncharacterized mislocalization artifact of Entacmaea quadricolor red fluorescent protein variants that is detectable at the single-molecule level in live Escherichia coli cells.

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Theory suggests that relatives will cooperate more, and compete less, because of an increased benefit for shared genes. In symbiotic partnerships, hosts may benefit from interacting with highly related symbionts because there is less conflict among the symbionts. This has been difficult to test empirically. We used the arbuscular mycorrhizal symbiosis to study the effects of fungal relatedness on host and fungal benefits, creating fungal networks varying in relatedness between two hosts, both in soil and in‐vitro. To determine how fungal relatedness affected overall transfer of nutrients, we fluorescently tagged phosphorus and quantified resource distribution between two root systems. We found that colonization by less‐related fungi was associated with increased fungal growth, lower transport of nutrients across the network, and lower plant benefit ‐ likely an outcome of increased fungal competition. More generally, we demonstrate how symbiont relatedness can mediate benefits of symbioses.