KidA, a multi-PAS domain protein, tunes the period of the cyanobacterial circadian oscillator.

The cyanobacterial clock presents a unique opportunity to understand the biochemical basis of circadian rhythms. The core oscillator, composed of the KaiA, KaiB, and KaiC proteins, has been extensively studied, but a complete picture of its connection to the physiology of the cell is lacking. To identify previously unknown components of the clock, we used KaiB locked in its active fold as bait in an immunoprecipitation/mass spectrometry approach. We found that the most abundant interactor, other than KaiC, was a putative diguanylate cyclase protein predicted to contain multiple Per-Arnt-Sim (PAS) domains, which we propose to name KidA. Here we show that KidA directly binds to the fold-switched active form of KaiB through its N-terminal PAS domains. We found that KidA shortens the period of the circadian clock both in vivo and in vitro and alters the ability of the clock to entrain to light-dark cycles. The dose-dependent effect of KidA on the clock period could be quantitatively recapitulated by a mathematical model in which KidA stabilizes the fold-switched form of KaiB, favoring rebinding to KaiC. Put together, our results show that the period and amplitude of the clock can be modulated by regulating the access of KaiB to the fold-switched form.

Ribosomally derived lipopeptides containing distinct fatty acyl moieties.

Lipopeptides represent a large group of microbial natural products that include important antibacterial and antifungal drugs and some of the most-powerful known biosurfactants. The vast majority of lipopeptides comprise cyclic peptide backbones N-terminally equipped with various fatty acyl moieties. The known compounds of this type are biosynthesized by nonribosomal peptide synthetases, giant enzyme complexes that assemble their products in a non-gene-encoded manner. Here, we report the genome-guided discovery of ribosomally derived, fatty-acylated lipopeptides, termed selidamides. Heterologous reconstitution of three pathways, two from cyanobacteria and one from an arctic, ocean-derived alphaproteobacterium, allowed structural characterization of the probable natural products and suggest that selidamides are widespread over various bacterial phyla. The identified representatives feature cyclic peptide moieties and fatty acyl units attached to (hydroxy)ornithine or lysine side chains by maturases of the GCN5-related N-acetyltransferase superfamily. In contrast to nonribosomal lipopeptides that are usually produced as congener mixtures, the three selidamides are selectively fatty acylated with C10, C12, or C16 fatty acids, respectively. These results highlight the ability of ribosomal pathways to emulate products with diverse, nonribosomal-like features and add to the biocatalytic toolbox for peptide drug improvement and targeted discovery.

The Emergence and Sustainability of Urban Entomology.

Urban entomology is the study of arthropod and other pests of the urban environment. It has gained worldwide recognition as a distinct discipline. Its origin is associated with Walter Ebeling's publication Urban Entomology in 1975. Urbanization, invasive pests, increased demand for pest management services, and changes in legislation collided in the 1970s to create a need for research and extension activities worldwide. This resulted in urban entomology as a discipline and, within two decades, its national and international recognition. In this review, we present the factors that led to the development of urban entomology and how they have shaped its current meaning. As urbanization intensifies and the global economy increases, the demands for urban pest management will continue to grow. We discuss how these future challenges may shape and alter the discipline.

The circadian clock ensures successful DNA replication in cyanobacteria.

Disruption of circadian rhythms causes decreased health and fitness, and evidence from multiple organisms links clock disruption to dysregulation of the cell cycle. However, the function of circadian regulation for the essential process of DNA replication remains elusive. Here, we demonstrate that in the cyanobacterium Synechococcus elongatus, a model organism with the simplest known circadian oscillator, the clock generates rhythms in DNA replication to minimize the number of open replication forks near dusk that would have to complete after sunset. Metabolic rhythms generated by the clock ensure that resources are available early at night to support any remaining replication forks. Combining mathematical modeling and experiments, we show that metabolic defects caused by clock-environment misalignment result in premature replisome disassembly and replicative abortion in the dark, leaving cells with incomplete chromosomes that persist through the night. Our study thus demonstrates that a major function of this ancient clock in cyanobacteria is to ensure successful completion of genome replication in a cycling environment.

Valhidepsin Lipopeptides from Chromobacterium vaccinii: Structures, Biosynthesis, and Coregulation with FR900359 Production.

Microbial secondary metabolites continue to provide a valuable source of both chemical matter and inspiration for drug discovery in a broad range of therapeutic areas. Beyond this, the corresponding microorganisms represent a sustainable modality for biotechnological production of structurally complex molecules at the quantities required for drug development or even commercial manufacturing. Chromobacterium vaccinii, which has recently been reported as a producer of the pharmacologically highly important Gq inhibitor FR900359 (FR), represents such an example. The characterization of an orphan biosynthetic gene cluster (BGC) located directly downstream of the frs BCG led to the discovery of eight new lipopeptides, valhidepsins A-H (1-8), produced by C. vaccinii. Their chemical structures were elucidated through analysis of 1D and 2D NMR data and high-resolution MS/MS fragmentation methods. The valhidepsins did not display significant antibiotic nor cytotoxic activities but showed surfactant properties. The cluster-compound correlation was demonstrated by generation of a knockout mutant, which abolished production of valhidepsins. This knockout mutant yielded a significantly increased isolated yield of FR.

A multiproducer microbiome generates chemical diversity in the marine sponge Mycale hentscheli.

Bacterial specialized metabolites are increasingly recognized as important factors in animal-microbiome interactions: for example, by providing the host with chemical defenses. Even in chemically rich animals, such compounds have been found to originate from individual members of more diverse microbiomes. Here, we identified a remarkable case of a moderately complex microbiome in the sponge host Mycale hentscheli in which multiple symbionts jointly generate chemical diversity. In addition to bacterial pathways for three distinct polyketide families comprising microtubule-inhibiting peloruside drug candidates, mycalamide-type contact poisons, and the eukaryotic translation-inhibiting pateamines, we identified extensive biosynthetic potential distributed among a broad phylogenetic range of bacteria. Biochemical data on one of the orphan pathways suggest a previously unknown member of the rare polytheonamide-type cytotoxin family as its product. Other than supporting a scenario of cooperative symbiosis based on bacterial metabolites, the data provide a rationale for the chemical variability of M. hentscheli and could pave the way toward biotechnological peloruside production. Most bacterial lineages in the compositionally unusual sponge microbiome were not known to synthesize bioactive metabolites, supporting the concept that microbial dark matter harbors diverse producer taxa with as yet unrecognized drug discovery potential.

Sustained order-disorder transitions in a model colloidal system driven by rhythmic crosslinking.

Biological systems have the unique ability to self-organize and generate autonomous motion and work. Motivated by this, we investigate a 2D model colloidal network that can repeatedly transition between disordered states of low connectivity and ordered states of high connectivity via rhythmic binding and unbinding of biomimetic crosslinkers. We use Langevin dynamics to investigate the time-dependent changes in structure and collective properties of this system as a function of colloidal packing fractions and crosslinker oscillation periods and characterize the degree of order in the system by using network connectivity, bond length distributions, and collective motion. Our simulations suggest that we can achieve distinct states of this colloidal system with pronounced differences in microstructural order and large residence times in the ordered state when crosslinker kinetics and lifetimes depend directly on the oscillation period and this oscillation period is much larger than the colloidal diffusion time. Our results will provide insights into the rational design of smart active materials that can independently cycle between ordered and disordered states with desired material properties on a programmed schedule.

Combined metabolic and target-site resistance mechanisms confer fipronil and deltamethrin resistance in field-collected German cockroaches (Blattodea: Ectobiidae).

Despite insecticide resistance issues, pyrethroids and fipronil have continued to be used extensively to control the German cockroach, Blattella germanica (L.) (Blattodea: Ectobiidae) for more than two decades. We evaluated the physiological insecticide resistance in five German cockroach populations collected from 2018 to 2020 and measured the extent of metabolic detoxification and target-site insensitivity resistance mechanisms. Topically applied doses of the 3 x LD95 of deltamethrin, fipronil, DDT, or dieldrin of a susceptible strain (UCR, Diagnostic Dose) failed to cause >23% mortality, and the 10 x LD95 of deltamethrin or fipronil failed to cause >53% mortality. All field-collected strains possessed a combination of metabolic and target-site insensitivity mechanisms that cause reduced susceptibility. Elevated activities of esterase and glutathione S-transferase were measured, and the synergists piperonyl butoxide or S,S,S-tributyl phosphorotrithioate increased topical mortality up to 100% for deltamethrin and 93% for fipronil 10 x LD95. The target-site mutations L993F of the para-homologous sodium channel and A302S of the GABA-gated chloride channel associated with pyrethroid and fipronil resistance, respectively, were found at ~80-100% frequency in field populations. Pyrethroid and fipronil spray formulations also were ineffective in a choice box assay against field-collected strains suggesting that these treatments would fail to control cockroaches under field conditions.

Modular Halogenation, α-Hydroxylation, and Acylation by a Remarkably Versatile Polyketide Synthase.

Bacterial multimodular polyketide synthases (PKSs) are large enzymatic assembly lines that synthesize many bioactive natural products of therapeutic relevance. While PKS catalysis is mostly based on fatty acid biosynthetic principles, polyketides can be further diversified by post-PKS enzymes. Here, we characterized a remarkably versatile trans-acyltransferase (trans-AT) PKS from Serratia that builds structurally complex macrolides via more than ten functionally distinct PKS modules. In the oocydin PKS, we identified a new oxygenation module that α-hydroxylates polyketide intermediates, a halogenating module catalyzing backbone γ-chlorination, and modular O-acetylation by a thioesterase-like domain. These results from a single biosynthetic assembly line highlight the expansive biochemical repertoire of trans-AT PKSs and provide diverse modular tools for engineered biosynthesis from a close relative of E. coli.

Bayesian modeling reveals metabolite-dependent ultrasensitivity in the cyanobacterial circadian clock.

Mathematical models can enable a predictive understanding of mechanism in cell biology by quantitatively describing complex networks of interactions, but such models are often poorly constrained by available data. Owing to its relative biochemical simplicity, the core circadian oscillator in Synechococcus elongatus has become a prototypical system for studying how collective dynamics emerge from molecular interactions. The oscillator consists of only three proteins, KaiA, KaiB, and KaiC, and near-24-h cycles of KaiC phosphorylation can be reconstituted in vitro. Here, we formulate a molecularly detailed but mechanistically naive model of the KaiA-KaiC subsystem and fit it directly to experimental data within a Bayesian parameter estimation framework. Analysis of the fits consistently reveals an ultrasensitive response for KaiC phosphorylation as a function of KaiA concentration, which we confirm experimentally. This ultrasensitivity primarily results from the differential affinity of KaiA for competing nucleotide-bound states of KaiC. We argue that the ultrasensitive stimulus-response relation likely plays an important role in metabolic compensation by suppressing premature phosphorylation at nighttime.

Roadmap on biology in time varying environments.

Biological organisms experience constantly changing environments, from sudden changes in physiology brought about by feeding, to the regular rising and setting of the Sun, to ecological changes over evolutionary timescales. Living organisms have evolved to thrive in this changing world but the general principles by which organisms shape and are shaped by time varying environments remain elusive. Our understanding is particularly poor in the intermediate regime with no separation of timescales, where the environment changes on the same timescale as the physiological or evolutionary response. Experiments to systematically characterize the response to dynamic environments are challenging since such environments are inherently high dimensional. This roadmap deals with the unique role played by time varying environments in biological phenomena across scales, from physiology to evolution, seeking to emphasize the commonalities and the challenges faced in this emerging area of research.

Automated structure prediction of trans-acyltransferase polyketide synthase products.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are among the most complex known enzymes from secondary metabolism and are responsible for the biosynthesis of highly diverse bioactive polyketides. However, most of these metabolites remain uncharacterized, since trans-AT PKSs frequently occur in poorly studied microbes and feature a remarkable array of non-canonical biosynthetic components with poorly understood functions. As a consequence, genome-guided natural product identification has been challenging. To enable de novo structural predictions for trans-AT PKS-derived polyketides, we developed the trans-AT PKS polyketide predictor (TransATor). TransATor is a versatile bio- and chemoinformatics web application that suggests informative chemical structures for even highly aberrant trans-AT PKS biosynthetic gene clusters, thus permitting hypothesis-based, targeted biotechnological discovery and biosynthetic studies. We demonstrate the applicative scope in several examples, including the characterization of new variants of bioactive natural products as well as structurally new polyketides from unusual bacterial sources.

Active cytoskeletal composites display emergent tunable contractility and restructuring.

The cytoskeleton is a model active matter system that controls processes as diverse as cell motility and mechanosensing. While both active actomyosin dynamics and actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay is lacking. Here, we couple microscale experiments with mechanistic modeling to elucidate how connectivity, rigidity, and force-generation affect emergent material properties in composite networks of actin, tubulin, and myosin. We use multi-spectral imaging, time-resolved differential dynamic microscopy and spatial image autocorrelation to show that ballistic contraction occurs in composites with sufficient flexibility and motor density, but that a critical fraction of microtubules is necessary to sustain controlled dynamics. The active double-network models we develop, which recapitulate our experimental findings, reveal that while percolated actomyosin networks are essential for contraction, only composites with comparable actin and microtubule densities can simultaneously resist mechanical stresses while supporting substantial restructuring. The comprehensive phase map we present not only provides important insight into the different routes the cytoskeleton can use to alter its dynamics and structure, but also serves as a much-needed blueprint for designing cytoskeleton-inspired materials that couple tunability with resilience and adaptability for diverse applications ranging from wound healing to soft robotics.

Molecular dynamics simulations of nucleotide release from the circadian clock protein KaiC reveal atomic-resolution functional insights.

The cyanobacterial clock proteins KaiA, KaiB, and KaiC form a powerful system to study the biophysical basis of circadian rhythms, because an in vitro mixture of the three proteins is sufficient to generate a robust ∼24-h rhythm in the phosphorylation of KaiC. The nucleotide-bound states of KaiC critically affect both KaiB binding to the N-terminal domain (CI) and the phosphotransfer reactions that (de)phosphorylate the KaiC C-terminal domain (CII). However, the nucleotide exchange pathways associated with transitions among these states are poorly understood. In this study, we integrate recent advances in molecular dynamics methods to elucidate the structure and energetics of the pathway for Mg·ADP release from the CII domain. We find that nucleotide release is coupled to large-scale conformational changes in the KaiC hexamer. Solvating the nucleotide requires widening the subunit interface leading to the active site, which is linked to extension of the A-loop, a structure implicated in KaiA binding. These results provide a molecular hypothesis for how KaiA acts as a nucleotide exchange factor. In turn, structural parallels between the CI and CII domains suggest a mechanism for allosteric coupling between the domains. We relate our results to structures observed for other hexameric ATPases, which perform diverse functions.

A Fundamental Unit of Cell Size in Bacteria.

A new study clarifies a relationship between growth, gene expression, and cell size in cyanobacteria. Quite unexpectedly, cyanobacteria and Escherichia coli appear to share an invariance principle to coordinate growth and chromosome replication. This principle allows quantitative predictions of cell size across a range of growth conditions in both organisms.

Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal network.

Actin and microtubule filaments, with their auxiliary proteins, enable the cytoskeleton to carry out vital processes in the cell by tuning the organizational and mechanical properties of the network. Despite their critical importance and interactions in cells, we are only beginning to uncover information about the composite network. The challenge is due to the high complexity of combining actin, microtubules, and their hundreds of known associated proteins. Here, we use fluorescence microscopy, fluctuation, and cross-correlation analysis to examine the role of actin and microtubules in the presence of an antiparallel microtubule crosslinker, MAP65, and a generic, strong actin crosslinker, biotin-NeutrAvidin. For a fixed ratio of actin and microtubule filaments, we vary the amount of each crosslinker and measure the organization and fluctuations of the filaments. We find that the microtubule crosslinker plays the principle role in the organization of the system, while, actin crosslinking dictates the mobility of the filaments. We have previously demonstrated that the fluctuations of filaments are related to the mechanics, implying that actin crosslinking controls the mechanical properties of the network, independent of the microtubule-driven re-organization.

Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton composites.

The cytoskeleton is able to precisely tune its structure and mechanics through interactions between semiflexible actin filaments, rigid microtubules and a suite of crosslinker proteins. However, the role that each of these components, as well as the interactions between them, plays in the dynamics of the composite cytoskeleton remains an open question. Here, we use optical tweezers microrheology and fluorescence confocal microscopy to reveal the surprising ways in which actin crosslinking tunes the viscoelasticity and mobility of actin-microtubule composites from steady-state to the highly nonlinear regime. While previous studies have shown that increasing crosslinking in actin networks increases elasticity and stiffness, we instead find that composite stiffness displays a striking non-monotonic dependence on actin crosslinking - first increasing then decreasing to a response similar to or even lower than un-linked composites. We further show that actin crosslinking has an unexpectedly strong impact on the mobility of microtubules; and it is in fact the microtubule mobility - dictated by crosslinker-driven rearrangements of actin filaments - that controls composite stiffness. This result is at odds with conventional thought that actin mobility drives cytoskeleton mechanics. More generally, our results demonstrate that - when crosslinking composite materials to confer strength and resilience - more is not always better.

Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break.

The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs). Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt) Rad51 filaments and also by one or more short Dmc1 filaments.

Mixtures of opposing phosphorylations within hexamers precisely time feedback in the cyanobacterial circadian clock.

Circadian oscillations are generated by the purified cyanobacterial clock proteins, KaiA, KaiB, and KaiC, through rhythmic interactions that depend on multisite phosphorylation of KaiC. However, the mechanisms that allow these phosphorylation reactions to robustly control the timing of oscillations over a range of protein stoichiometries are not clear. We show that when KaiC hexamers consist of a mixture of differentially phosphorylated subunits, the two phosphorylation sites have opposing effects on the ability of each hexamer to bind to the negative regulator KaiB. We likewise show that the ability of the positive regulator KaiA to act on KaiC depends on the phosphorylation state of the hexamer and that KaiA and KaiB recognize alternative allosteric states of the KaiC ring. Using mathematical models with kinetic parameters taken from experimental data, we find that antagonism of the two KaiC phosphorylation sites generates an ultrasensitive switch in negative feedback strength necessary for stable circadian oscillations over a range of component concentrations. Similar strategies based on opposing modifications may be used to support robustness in other timing systems and in cellular signaling more generally.

Costs of Clock-Environment Misalignment in Individual Cyanobacterial Cells.

Circadian rhythms are endogenously generated daily oscillations in physiology that are found in all kingdoms of life. Experimental studies have shown that the fitness of Synechococcus elongatus, a photosynthetic microorganism, is severely affected in non-24-h environments. However, it has been difficult to study the effects of clock-environment mismatch on cellular physiology because such measurements require a precise determination of both clock state and growth rate in the same cell. Here, we designed a microscopy platform that allows us to expose cyanobacterial cells to pulses of light and dark while quantitatively measuring their growth, division rate, and circadian clock state over many days. Our measurements reveal that decreased fitness can result from a catastrophic growth arrest caused by unexpected darkness in a small subset of cells with incorrect clock times corresponding to the subjective morning. We find that the clock generates rhythms in the instantaneous growth rate of the cell, and that the time of darkness vulnerability coincides with the time of most rapid growth. Thus, the clock mediates a fundamental trade-off between growth and starvation tolerance in cycling environments. By measuring the response of the circadian rhythm to dark pulses of varying lengths, we constrain a mathematical model of a population's fitness under arbitrary light/dark schedules. This model predicts that the circadian clock is only advantageous in highly regular cycling environments with frequencies sufficiently close to the natural frequency of the clock.