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Transposases drive chromosomal rearrangements and the dissemination of drug-resistance genes and toxins1–3. Although some transposases act alone, many rely on dedicated AAA+ ATPase subunits that regulate site selectivity and catalytic function through poorly understood mechanisms. Using IS21 as a model transposase system, we show how an ATPase regulator uses nucleotide-controlled assembly and DNA deformation to enable structure-based site selectivity, transposase recruitment, and activation and integration. Solution and cryogenic electron microscopy studies show that the IstB ATPase self-assembles into an autoinhibited pentamer of dimers that tightly curves target DNA into a half-coil. Two of these decamers dimerize, which stabilizes the target nucleic acid into a kinked S-shaped configuration that engages the IstA transposase at the interface between the two IstB oligomers to form an approximately 1 MDa transpososome complex. Specific interactions stimulate regulator ATPase activity and trigger a large conformational change on the transposase that positions the catalytic site to perform DNA strand transfer. These studies help explain how AAA+ ATPase regulators—which are used by classical transposition systems such as Tn7, Mu and CRISPR-associated elements—can remodel their substrate DNA and cognate transposases to promote function.Solution and cryogenic electron microscopy studies using IS21 as a model transposase system show how AAA+ ATPases induce structural changes to prime target DNA and activate their associated transposases. [ABSTRACT FROM AUTHOR]

Transposases are ubiquitous enzymes that catalyze DNA rearrangement events with broad impacts on gene expression, genome evolution, and the spread of drug-resistance in bacteria. Here, we use biochemical and structural approaches to define the molecular determinants by which IstA, a transposase present in the widespread IS21 family of mobile elements, catalyzes efficient DNA transposition. Solution studies show that IstA engages the transposon terminal sequences to form a high-molecular weight complex and promote DNA integration. A 3.4 Å resolution structure of the transposase bound to transposon ends corroborates our biochemical findings and reveals that IstA self-assembles into a highly intertwined tetramer that synapses two supercoiled terminal inverted repeats. The three-dimensional organization of the IstA•DNA cleaved donor complex reveals remarkable similarities with retroviral integrases and classic transposase systems, such as Tn7 and bacteriophage Mu, and provides insights into IS21 transposition. Spínola-Amilibia et al. present the cryo-EM structure of the IS21 transposase in complex with the donor DNA and show that IstA recognizes the transposon ends with a highly intertwined configuration to facilitate the strand-transfer reaction. [ABSTRACT FROM AUTHOR]

The global spread of bacterial antimicrobial resistance is associated to millions of deaths from bacterial infections per year, many of which were previously treatable. This, combined with slow antibiotic deployment, has created an urgent need for developing new antibiotics. A still clinically unexploited mode of action consists in suppressing bacterial cell division. FtsZ, an assembling GTPase, is the key protein organizing division in most bacteria and an attractive target for antibiotic discovery. Nevertheless, developing effective antibacterial inhibitors targeting FtsZ has proven challenging. Here we review our decade-long multidisciplinary research on small molecule inhibitors of bacterial division, in the context of global efforts to discover FtsZ-targeting antibiotics. We focus on methods to characterize synthetic inhibitors that either replace bound GTP from the FtsZ nucleotide binding pocket conserved across diverse bacteria or selectively bind into the allosteric site at the interdomain cleft of FtsZ from Bacillus subtilis and the pathogen Staphylococcus aureus. These approaches include phenotype screening combined with fluorescence polarization screens for ligands binding into each site, followed by detailed cytological profiling, and biochemical and structural studies. The results are analyzed to design an optimized workflow to identify effective FtsZ inhibitors, and new approaches for the discovery of FtsZ-targeting antibiotics are discussed. [ABSTRACT FROM AUTHOR]

The essential bacterial division protein FtsZ uses GTP binding and hydrolysis to assemble into dynamic filaments that treadmill around the Z‐ring, guiding septal wall synthesis and cell division. FtsZ is a structural homolog of tubulin and a target for discovering new antibiotics. Here, using FtsZ from the pathogen S. aureus (SaFtsZ), we reveal that, prior to assembly, FtsZ monomers require nucleotide binding for folding; this is possibly relevant to other mesophilic FtsZs. Apo‐SaFtsZ is essentially unfolded, as assessed by nuclear magnetic resonance and circular dichroism. Binding of GTP (≥ 1 mm) dramatically shifts the equilibrium toward the active folded protein. Supportingly, SaFtsZ refolded with GDP crystallizes in a native structure. Apo‐SaFtsZ also folds with 3.4 m glycerol, enabling high‐affinity GTP binding (KD 20 nm determined by isothermal titration calorimetry) similar to thermophilic stable FtsZ. Other stabilizing agents that enhance nucleotide binding include ethylene glycol, trimethylamine N‐oxide, and several bacterial osmolytes. High salt stabilizes SaFtsZ without bound nucleotide in an inactive twisted conformation. We identified a cavity behind the SaFtsZ‐GDP nucleotide‐binding pocket that harbors different small compounds, which is available for extended nucleotide‐replacing inhibitors. Furthermore, we devised a competition assay to detect any inhibitors that overlap the nucleotide site of SaFtsZ, or Escherichia coli FtsZ, employing osmolyte‐stabilized apo‐FtsZs and the specific fluorescence anisotropy change in mant‐GTP upon dissociation from the protein. This robust assay provides a basis to screening for high‐affinity GTP‐replacing ligands, which combined with structural studies and phenotypic profiling should facilitate development of a next generation of FtsZ‐targeting antibacterial inhibitors. [ABSTRACT FROM AUTHOR]

Summary: Cell division in most bacteria is directed by FtsZ, a conserved tubulin‐like GTPase that assembles forming the cytokinetic Z‐ring and constitutes a target for the discovery of new antibiotics. The developmental regulator MciZ, a 40‐amino acid peptide endogenously produced during Bacillus subtilis sporulation, halts cytokinesis in the mother cell by inhibiting FtsZ. The crystal structure of a FtsZ:MciZ complex revealed that bound MciZ extends the C‐terminal β‐sheet of FtsZ blocking its assembly interface. Here we demonstrate that exogenously added MciZ specifically inhibits B. subtilis cell division, sporulation and germination, and provide insight into MciZ molecular recognition by FtsZ from different bacteria. MciZ and FtsZ form a complex with sub‐micromolar affinity, analyzed by analytical ultracentrifugation, laser biolayer interferometry and isothermal titration calorimetry. Synthetic MciZ analogs, carrying single amino acid substitutions impairing MciZ β‐strand formation or hydrogen bonding to FtsZ, show a gradual reduction in affinity that resembles their impaired activity in bacteria. Gene sequences encoding MciZ spread across genus Bacillus and synthetic MciZ slows down cell division in Bacillus species, including pathogenic Bacillus cereus and Bacillus anthracis. Moreover, B. subtilis MciZ is recognized by the homologous FtsZ from Staphylococcus aureus and inhibits division when it is expressed into S. aureus cells. Synthetic peptide MciZ is a specific inhibitor of B. subtilis division, sporulation and germination that binds with high affinity to key cell division protein FtsZ, blocking its assembly and the formation of the cytokinetic Z‐ring. MciZ activity extends to other Bacillus, including pathogenic B. cereus – anthracis. MciZ is also recognized by FtsZ from S. aureus and intracellularly expressed MiciZ inhibits S. aureus division. Our results suggest the possibility of designing MciZ‐based peptide antibacterials. [ABSTRACT FROM AUTHOR]

Cell division protein FtsZ is the organizer of the cytokinetic ring in almost all bacteria and a target for the discovery of new antibacterial agents that are needed to counter widespread antibiotic resistance. Bacterial cytological profiling, using quantitative microscopy, is a powerful approach for identifying the mechanism of action of antibacterial molecules affecting different cellular pathways. We have determined the cytological profile on Bacillus subtilis cells of a selection of small molecule inhibitors targeting FtsZ on different binding sites. FtsZ inhibitors lead to long undivided cells, impair the normal assembly of FtsZ into the midcell Z-rings, induce aberrant ring distributions, punctate FtsZ foci, membrane spots and also modify nucleoid length. Quantitative analysis of cell and nucleoid length combined, or the Z-ring distribution, allows categorizing FtsZ inhibitors and to distinguish them from antibiotics with other mechanisms of action, which should be useful for identifying new antibacterial FtsZ inhibitors. Biochemical assays of FtsZ polymerization and GTPase activity combined explain the cellular effects of the FtsZ polymer stabilizing agent PC190723 and its fragments. MciZ is a 40-aminoacid endogenous inhibitor of cell division normally expressed during sporulation in B. subtilis. Using FtsZ cytological profiling we have determined that exogenous synthetic MciZ is an effective inhibitor of B. subtilis cell division, Z-ring formation and localization. This finding supports our cell-based approach to screen for FtsZ inhibitors and opens new possibilities for peptide inhibitors of bacterial cell division. [ABSTRACT FROM AUTHOR]

The increasing use of the 7-valent pneumococcal conjugate vaccine has been accompanied by the rise of non-vaccine serotypes colonizing the human nasopharynx. The vast majority of infections are caused by microorganisms that grow in biofilms. It has recently been shown that the formation of S treptococcus pneumoniae biofilms in vivo and in vitro is hindered by the presence of capsular polysaccharide. The biofilm-forming capacity of pneumococcal clinical isolates with different types of capsular polysaccharide and various isogenic transformants was examined. Strains of serotypes 19 A and 19 F, but not 19 B and 19 C, formed ≥ 80% of the quantity of biofilm associated with a non-encapsulated control strain. Strains of serogroup 6 also showed significant biofilm-forming capacity. The capsules of serotypes 19A and 19F, and serogroup 6 contain the disaccharides α-D-Glc p-(1→2)- α-L-Rha p-(1→ and α-D-Glc p-(1→3)- α-L-Rha p-(1→. Serotype 18 A and serotypes 18 B/18 C have very similar capsular disaccharides: α-D-Glc pNAc-(1→3)- β-L-Rha p-(1→ and α-D-Glc p-(1→3)- β-L-Rha p-(1→ respectively. However, the strains of serogroup 18 showed impaired biofilm formation. These results indicate that the chemical composition/structure of the capsular polysaccharide is crucial to the biofilm-forming capacity of pneumococcal serotypes. Testing of the in vitro biofilm-forming ability of isogenic transformants expressing different capsular polysaccharides may help predict the emergence of colonizing, non-vaccine serotypes. [ABSTRACT FROM AUTHOR]

Conidiophore formation in Aspergillus nidulans involves a developmental programme in which vegetative hyphae give rise to an ordered succession of differentiated cells: foot cell, stalk, vesicle, metulae, phialides and conidia. The developmental transition requires factors that are expressed in vegetative hyphae that activate the expression of the main regulator of conidiation, BrlA. One such element is the bZIP-type transcription factor FlbB. We found that flbB- mutants show defective branching patterns and are susceptible to autolysis under high sorbitol or sucrose concentrations, revealing a role in vegetative growth. In addition, FlbB plays a role in conidiophore initiation, as its upregulation reduces conidiophore vesicle swelling and generates a reduced number of metulae. FlbB was located at the tip of growing metulae, following a similar pattern as described in vegetative hyphae. In wild-type strains, the transition from metulae to phialides could be reversed to generate vegetative hyphae, indicating the existence of a specific control point at this stage of conidiophore formation. The combined evidence points to FlbB as a key factor in the transition to asexual development, playing a role at various control points in which the process could be reversed. [ABSTRACT FROM AUTHOR]

The veA gene is a light-dependent regulator governing development and secondary metabolism in Aspergillus nidulans. We have identified a putative bipartite nuclear localization signal (NLS) motif in the A. nidulans VeA amino acid sequence and demonstrated its functionality when expressed in yeast. Furthermore, migration of VeA to the nucleus was dependent on the importin α. This bipartite NLS is also functional when VeA is expressed in A. nidulans. Interestingly, we found that VeA migration to the nucleus is light-dependent. While in the dark VeA is located mainly in the nuclei, under light VeA is found abundantly in the cytoplasm. The VeA1 mutant protein (lacking the first 36 amino acids at the N-terminus) was found predominantly in the cytoplasm independent of illumination. This indicates that the truncated bipartite NLS in VeA1 is not functional and fails to respond to light. These results might explain the lack of the morphological light-dependent response in strains carrying the veA1 allele. We also evaluated the effect of light on production of the mycotoxin sterigmatocystin in a veA wild-type and the veA1 mutant strains and found that the highest amount of toxin was produced by the veA+ strain growing in the dark, condition favouring accumulation of VeA in the nucleus. [ABSTRACT FROM AUTHOR]

Karyopherins are transporters involved in the bidirectional, selective and active transport of macromolecules through nuclear pores. Importin-β1 is the paradigm of karyopherins and, together with its cargo-adapter importin-α, mediates the general nuclear import pathway. Here we show the existence of different cellular pools of both importin-α and -β1 homologues, KapA and KapB, in the coenocytic ascomycete Aspergillus nidulans. Fluorescence analysis of haploid and diploid strains expressing KapB::GFP and/or KapA::mRFP showed patches of both karyopherins concurrently translocating long distances in apically-growing cells. Anterograde and retrograde movements allowed those patches to reach cell tips and distal regions with an average speed in the range of μm/s. This bidirectional traffic required microtubules as well as kinesin and dynein motors, since it is blocked by benomyl and also by the inactivation of the dynein/dynactin complex through nudA1 or nudK317 mutations. Deletion of Kinesin-3 motor UncA, required for the transport through detyrosinated microtubules, strongly inhibited KapA and KapB movement along hyphae. Overall, this is the first report describing the bidirectional dynamics of the main nuclear import system in coenocytic fungi. A functional link is proposed between two key cellular machines of the filamentous fungal cell: nuclear transport and the tip-growth apparatus. [ABSTRACT FROM AUTHOR]