Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis.

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference of Bacillus subtilis cells for minutes-long periods using single-molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single-molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes associated with the MreB filament. Altogether these results demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.

Peptidoglycan synthesis drives a single population of septal cell wall synthases during division in Bacillus subtilis.

Bacterial cell division requires septal peptidoglycan (sPG) synthesis by the divisome complex. Treadmilling of the essential tubulin homologue FtsZ has been implicated in septal constriction, though its precise role remains unclear. Here we used live-cell single-molecule imaging of the divisome transpeptidase PBP2B to investigate sPG synthesis dynamics in Bacillus subtilis. In contrast to previous models, we observed a single population of processively moving PBP2B molecules whose motion is driven by peptidoglycan synthesis and is not associated with FtsZ treadmilling. However, despite the asynchronous motions of PBP2B and FtsZ, a partial dependence of PBP2B processivity on FtsZ treadmilling was observed. Additionally, through single-molecule counting experiments we provide evidence that the divisome synthesis complex is multimeric. Our results support a model for B. subtilis division where a multimeric synthesis complex follows a single track dependent on sPG synthesis whose activity and dynamics are asynchronous with FtsZ treadmilling.

A new role for monomeric ParA/Soj in chromosome dynamics in Bacillus subtilis.

ParABS (Soj-Spo0J) systems were initially implicated in plasmid and chromosome segregation in bacteria. However, it is now increasingly understood that they play multiple roles in cell cycle events in Bacillus subtilis, and possibly other bacteria. In a recent study, monomeric forms of ParA/Soj have been implicated in regulating aspects of chromosome dynamics during B. subtilis sporulation. In this commentary, I will discuss the known roles of ParABS systems, explore why sporulation is a valuable model for studying these proteins, and the new insights into the role of monomeric ParA/Soj. Finally, I will touch upon some of the future work that remains.

Chromosome remodelling by SMC/Condensin in B. subtilis is regulated by monomeric Soj/ParA during growth and sporulation.

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving adenosine triphosphate (ATP)-dependent dimerization and DNA binding, facilitating chromosome segregation in bacteria. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. Here, we show that a major redistribution of SMC complexes drives axial filament formation in a process regulated by ParA/Soj. Furthermore, and unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyze ATP. These results reveal additional roles for ParA/Soj proteins in the regulation of SMC dynamics in bacteria and yet further complexity in the web of interactions involving chromosome replication, segregation and organization, controlled by ParAB and SMC.

Characterization of the L-form switch in the Gram-negative pathogen Streptobacillus moniliformis.

Almost all major classes of bacteria are surrounded by a peptidoglycan cell wall, which is a crucial target for antibiotics. It is now understood that many bacteria can tolerate loss of the cell wall provided that they are in an isotonic environment. Furthermore, in some cases the cells can continue to proliferate in a state known as the L-form. L-form proliferation occurs by an unusual blebbing or tubulation mechanism that is completely independent of the normally essential division machine or cell wall synthetic enzymes, and is resistant to cell wall-active antibiotics. However, the growth is limited by reactive oxygen species generated by the respiratory chain pathway. In this work, we examined the walled to L-form transition in a pathogenic Gram-negative bacterium, Streptobacillus moniliformis, which naturally lacks the respiratory chain pathway, under aerobic conditions. L-form-like cells often emerged spontaneously, but proliferation was not observed unless the cells were treated with cell wall-active antibiotics. Time-lapse imaging revealed that cell division of S. moniliformis L-forms involves unusual membrane dynamics with an apparent imbalance between outer membrane and cytoplasmic volume growth. The results suggest that outer membrane expansion may be an important general factor for L-form proliferation of diderm bacteria.

Genetic variability of Arthurdendyus triangulatus (Dendy, 1894), a non-native invasive land planarian.

Arthurdendyus triangulatus (Dendy, 1894) is a land planarian native to New Zealand which has become established in the United Kingdom and the Faroe Islands during the last 60 years. The species has become prevalent and widely established in Scotland mediated by human activity mostly through the exchange of plants and associated soil. As a predator of earthworms, concerns regarding both the direct impact on earthworm abundance and diversity and the indirect impact on those birds and mammals that have earthworms as a primary dietary component led to A. triangulatus being the subject of both national and EU regulation. Whilst much is known regarding the ecology of A. triangulatus there is a significant knowledge gap regarding the genetic variability of the species. Using four DNA target regions cytochrome oxidase (CoI), elongation factor (EF), internal transcribed spacer region (ITS-1) and large subunit (LSU), we characterised the genetic variability of A. triangulatus populations across its full geographic range in Scotland and included a few populations from New Zealand, England and Northern Ireland.                Two DNA regions, ITS-1 and CoI, revealed inter-population variability yielding well supported genetic structure in predominantly Scottish populations. We also identified considerable intra-specific and intra-individual heterogeneity in both ribosomal and mitochondrial regions, including the prevalence of pseudo-gene nuclear encoded mitochondrial DNA (Numt), the latter not previously reported for Platyhelminthes. Furthermore, given the presence of multiple ITS-1 haplotypes in individual specimens of A. triangulatus it is not possible to make definitive comment to support previously published findings that A. triangulatus was subjected to multiple introductions into the UK.

Impact of land use and management practices on soil nematode communities of Machair, a low-input calcareous ecosystem of conservation importance.

Machair is a vulnerable low-lying coastal ecosystem with internationally recognised conservation importance. It is characterised by wind-blown calcareous shell-sand soils that support a patchwork of low-input land-use types including species rich grasslands and small-scale arable production. In contrast to numerous above-ground studies, few below-ground studies have been made on the Machair. Thus, a knowledge gap exists, and no baseline data is available to determine the impact, if any, of fundamental changes in Machair land management practices such as a move from traditional rotational to permanent grazing, and increased use of inorganic fertiliser. To address this knowledge deficit, we assessed the impact of different agronomic management practices (cropped, fallow and grasslands) on the structure of soil nematode communities over a two-year period along a geographically limited north-south gradient of coastal Machair of the Outer Hebrides archipelago. Land use followed by season were the main drivers of nematode communities from Machair soils. Functionally, nematode communities from grassland were typically distinct from cropped or fallow communities driven primarily by differential contributions to the overall nematode community by the dominant bacterial-feeding nematodes. Temporally, nematode communities sampled in spring and autumn were distinct.

A high-throughput whole cell screen to identify inhibitors of Mycobacterium tuberculosis.

Tuberculosis is a disease of global importance for which novel drugs are urgently required. We developed a whole-cell phenotypic screen which can be used to identify inhibitors of Mycobacterium tuberculosis growth. We used recombinant strains of virulent M. tuberculosis which express far-red fluorescent reporters and used fluorescence to monitor growth in vitro. We optimized our high throughput assays using both 96-well and 384-well plates; both formats gave assays which met stringent reproducibility and robustness tests. We screened a compound set of 1105 chemically diverse compounds previously shown to be active against M. tuberculosis and identified primary hits which showed ≥ 90% growth inhibition. We ranked hits and identified three chemical classes of interest-the phenoxyalkylbenzamidazoles, the benzothiophene 1-1 dioxides, and the piperidinamines. These new compound classes may serve as starting points for the development of new series of inhibitors that prevent the growth of M. tuberculosis. This assay can be used for further screening, or could easily be adapted to other strains of M. tuberculosis.

Parallel Microbial Ecology of Pasteuria and Nematode Species in Scottish Soils.

Pasteuria spp. are endospore forming bacteria which act as natural antagonists to many of the most economically significant plant parasitic nematodes (PPNs). Highly species-specific nematode suppression may be observed in soils containing a sufficiently high density of Pasteuria spp. spores. This suppression is enacted by the bacteria via inhibition of root invasion and sterilization of the nematode host. Molecular methods for the detection of Pasteuria spp. from environmental DNA (eDNA) have been described; however, these methods are limited in both scale and in depth. We report the use of small subunit rRNA gene metabarcoding to profile Pasteuria spp. and nematode communities in parallel. We have investigated Pasteuria spp. population structure in Scottish soils using eDNA from two sources: soil extracted DNA from the second National Soil Inventory of Scotland (NSIS2); and nematode extracted DNA collected from farms in the East Scotland Farm Network (ESFN). We compared the Pasteuria spp. community culture to both nematode community structure and the physiochemical properties of soils. Our results indicate that Pasteuria spp. populations in Scottish soils are broadly dominated by two sequence variants. The first of these aligns with high identity to Pasteuria hartismeri, a species first described parasitizing Meloidogyne ardenensis, a nematode parasite of woody and perennial plants in northern Europe. The second aligns with a Pasteuria-like sequence which was first recovered from a farm near Edinburgh which was found to contain bacterial feeding nematodes and Pratylenchus spp. encumbered by Pasteuria spp. endospores. Further, soil carbon, moisture, bulk density, and pH showed a strong correlation with the Pasteuria spp. community composition. These results indicate that metabarcoding is appropriate for the sensitive, specific, and semi-quantitative profiling of Pasteuria species from eDNA.

Identification of Enolase as the Target of 2-Aminothiazoles in Mycobacterium tuberculosis.

Tuberculosis is a massive global burden and Mycobacterium tuberculosis is increasingly resistant to first- and second-line drugs. There is an acute need for new anti-mycobacterial drugs with novel targets. We previously evaluated a series of 2-aminothiazoles with activity against Mycobacterium tuberculosis. In this study, we identify the glycolytic enzyme enolase as the target of these molecules using pull down studies. We demonstrate that modulation of the level of enolase expression affects sensitivity to 2-aminothiazoles; increased expression leads to resistance while decreased protein levels increase sensitivity. Exposure to 2-aminothiazoles results in increased levels of metabolites preceding the action of enolase in the glycolytic pathway and decreased ATP levels. We demonstrate that 2-aminothiazoles inhibit the activity of the human α-enolase, which could also account for the cytotoxicity of some of those molecules. If selectivity for the bacterial enzyme over the human enzyme could be achieved, enolase would represent an attractive target for M. tuberculosis drug discovery and development efforts.

Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo.

Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity.

A mechanism for FtsZ-independent proliferation in Streptomyces.

The central player in bacterial cell division, FtsZ, is essential in almost all organisms in which it has been tested, with the most notable exception being Streptomyces. Streptomycetes differ from many bacteria in growing from the cell tip and undergoing branching, similar to filamentous fungi. Here we show that limited cell damage, either mechanical or enzymatic, leads to near complete destruction of mycelial microcolonies of a Streptomyces venezuelae ftsZ mutant. This result is consistent with a lack of ftsZ-dependent cross-walls and may be inconsistent with a recently proposed role for membrane structures in the proliferation of ftsZ mutants in other Streptomyces species. Rare surviving fragments of mycelium, usually around branches, appear to be the preferred sites of resealing. Restoration of growth in hyphal fragments of both wild-type and ftsZ mutant hyphae can occur at multiple sites, via branch-like outgrowths containing DivIVA protein at their tips. Thus, our results highlight branching as a means of FtsZ-independent cell proliferation.

The Scaffolding Protein IQGAP1 Interacts with NLRC3 and Inhibits Type I IFN Production.

Sensing of cytosolic nucleotides is a critical initial step in the elaboration of type I IFN. One of several upstream receptors, cyclic GMP-AMP synthase, binds to cytosolic DNA and generates dicyclic nucleotides that act as secondary messengers. These secondary messengers bind directly to stimulator of IFN genes (STING). STING recruits TNFR-associated NF-κB kinase-binding kinase 1 which acts as a critical node that allows for efficient activation of IFN regulatory factors to drive the antiviral transcriptome. NLRC3 is a recently characterized nucleotide-binding domain, leucine-rich repeat containing protein (NLR) that negatively regulates the type I IFN pathway by inhibiting subcellular redistribution and effective signaling of STING, thus blunting the transcription of type I IFNs. NLRC3 is predominantly expressed in lymphoid and myeloid cells. IQGAP1 was identified as a putative interacting partner of NLRC3 through yeast two-hybrid screening. In this article, we show that IQGAP1 associates with NLRC3 and can disrupt the NLRC3-STING interaction in the cytosol of human epithelial cells. Furthermore, knockdown of IQGAP1 in THP1 and HeLa cells causes significantly more IFN-ß production in response to cytosolic nucleic acids. This result phenocopies NLRC3-deficient macrophages and fibroblasts and short hairpin RNA knockdown of NLRC3 in THP1 cells. Our findings suggest that IQGAP1 is a novel regulator of type I IFN production, possibly via interacting with NLRC3 in human monocytic and epithelial cells.

A high content microscopy assay to determine drug activity against intracellular Mycobacterium tuberculosis.

Tuberculosis is one of the infectious diseases with the greatest global burden, affecting millions of people. The rise of multi- and extensively-drug resistant forms of Mycobacterium tuberculosis over the last few decades has highlighted the urgent need for development of new drugs to treat the disease. Many drug development pipelines are based on in vitro assays examining a compound's effect on M. tuberculosis alone. These do not account for the effect of a compound on mammalian cells nor the interaction between host and pathogen. We therefore developed a live-cell fluorescence-based screen utilizing high content microscopy of mammalian macrophages infected with M. tuberculosis to screen for compounds with both substantial inhibition of M. tuberculosis growth and low cytotoxicity. Isoniazid, a first line tuberculosis drug, and staurosporine, a compound with well documented cytotoxic activity, were used to validate the assay. These and other control compounds showed results for M. tuberculosis growth consistent with the field. Together, this method of screening allows for high throughput testing of potential tuberculosis drugs while capturing more information per compound in a physiologically relevant context.

Imidazoles Induce Reactive Oxygen Species in Mycobacterium tuberculosis Which Is Not Associated with Cell Death.

Azoles are a class of antimicrobial drugs used clinically to treat yeast and fungal infections. Against pathogenic yeast and fungi, azoles act by inhibiting the activity of the cytochrome P450 Cyp51, which is involved in the synthesis of a critical component of the yeast and fungal cell membrane. Azoles have antibacterial activity, including against mycobacteria, but the basis for this activity is not well-understood. We demonstrated that imidazoles are bactericidal to Mycobacterium tuberculosis. A marked increase in reactive oxygen species (ROS) was observed within imidazole-treated M. tuberculosis. The generation of ROS did not appear to be related to the mechanism of killing of imidazoles, as the addition of antioxidants or altered expression of detoxifying enzymes had no effect on growth. We examined the metabolic changes induced by econazole treatment in both wild-type and econazole-resistant mutant strains of M. tuberculosis. Econazole treatment induced changes in carbohydrates, amino acids, and energy metabolism in both strains. Notably, the untreated mutant strain had a metabolic profile similar to the wild-type drug-treated cells, suggesting that adaptation to similar stresses may play a role in econazole resistance.

Complex polar machinery required for proper chromosome segregation in vegetative and sporulating cells of Bacillus subtilis.

Chromosome segregation is an essential process of cell multiplication. In prokaryotes, segregation starts with the newly replicated sister origins of replication, oriCs, which move apart to defined positions in the cell. We have developed a genetic screen to identify mutants defective in placement of oriC during spore development in the Gram-positive bacterium Bacillus subtilis. In addition to the previously identified proteins Soj and DivIVA, our screen identified several new factors involved in polar recruitment of oriC: a reported regulator of competence ComN, and the regulators of division site selection MinD and MinJ. Previous work implicated Soj as an important regulator of oriC positioning in the cell. Our results suggest a model in which the DivIVA-interacting proteins ComN and MinJ recruit MinD to the cell pole, and that these proteins work upstream of Soj to enable oriC placement. We show that these proteins form a polar complex, which acts in parallel with but distinct from the sporulation-specific RacA pathway of oriC placement, and also functions during vegetative growth. Our study further shows that MinD has two distinct cell cycle roles, in cell division and chromosome segregation, and highlights that cell probably use multiple parallel mechanisms to ensure accurate chromosome segregation.

The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the ß-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2.

Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein ß-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the ß-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate ß-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases ß-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy.

Genetics on the Fly: A Primer on the Drosophila Model System.

Fruit flies of the genus Drosophila have been an attractive and effective genetic model organism since Thomas Hunt Morgan and colleagues made seminal discoveries with them a century ago. Work with Drosophila has enabled dramatic advances in cell and developmental biology, neurobiology and behavior, molecular biology, evolutionary and population genetics, and other fields. With more tissue types and observable behaviors than in other short-generation model organisms, and with vast genome data available for many species within the genus, the fly's tractable complexity will continue to enable exciting opportunities to explore mechanisms of complex developmental programs, behaviors, and broader evolutionary questions. This primer describes the organism's natural history, the features of sequenced genomes within the genus, the wide range of available genetic tools and online resources, the types of biological questions Drosophila can help address, and historical milestones.

The two SAMP repeats and their phosphorylation state in Drosophila Adenomatous polyposis coli-2 play mechanistically distinct roles in negatively regulating Wnt signaling.

The tumor suppressor Adenomatous polyposis coli (APC) plays a key role in regulating the canonical Wnt signaling pathway as an essential component of the ß-catenin destruction complex. C-terminal truncations of APC are strongly implicated in both sporadic and familial forms of colorectal cancer. However, many questions remain as to how these mutations interfere with APC's tumor suppressor activity. One set of motifs frequently lost in these cancer-associated truncations is the SAMP repeats that mediate interactions between APC and Axin. APC proteins in both vertebrates and Drosophila contain multiple SAMP repeats that lack high sequence conservation outside of the Axin-binding motif. In this study, we tested the functional redundancy between different SAMPs and how these domains are regulated, using Drosophila APC2 and its two SAMP repeats as our model. Consistent with sequence conservation-based predictions, we show that SAMP2 has stronger binding activity to Axin in vitro, but SAMP1 also plays an essential role in the Wnt destruction complex in vivo. In addition, we demonstrate that the phosphorylation of SAMP repeats is a potential mechanism to regulate their activity. Overall our findings support a model in which each SAMP repeat plays a mechanistically distinct role but they cooperate for maximal destruction complex function.