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Iron-sulfur clusters function as cofactors of a wide range of proteins, with diverse molecular roles in both prokaryotic and eukaryotic cells. Dedicated machineries assemble the clusters and deliver them to the final acceptor molecules in a tightly regulated process. In the prototypical Gram-negative bacterium Escherichia coli , the two existing iron-sulfur cluster assembly systems, iron-sulfur cluster (ISC) and sulfur assimilation (SUF) pathways, are closely interconnected. The ISC pathway regulator, IscR, is a transcription factor of the helix-turn-helix type that can coordinate a [2Fe-2S] cluster. Redox conditions and iron or sulfur availability modulate the ligation status of the labile IscR cluster, which in turn determines a switch in DNA sequence specificity of the regulator: cluster-containing IscR can bind to a family of gene promoters (type-1) whereas the clusterless form recognizes only a second group of sequences (type-2). However, iron-sulfur cluster biogenesis in Gram-positive bacteria is not so well characterized, and most organisms of this group display only one of the iron-sulfur cluster assembly systems. A notable exception is the unique Gram-positive dissimilatory metal reducing bacterium Thermincola potens , where genes from both systems could be identified, albeit with a diverging organization from that of Gram-negative bacteria. We demonstrated that one of these genes encodes a functional IscR homolog and is likely involved in the regulation of iron-sulfur cluster biogenesis in T. potens . Structural and biochemical characterization of T. potens and E. coli IscR revealed a strikingly similar architecture and unveiled an unforeseen conservation of the unique mechanism of sequence discrimination characteristic of this distinctive group of transcription regulators.

Exopolysaccharides and extracellular DNA are important structural components that contribute to the self-assembly of large aggregates or microcolonies that are characteristic of biofilms. Pseudomonas aeruginosa is capable of producing multiple exopolysaccharides, including alginate, Psl, and Pel. At present, little is known about Pel’s chemical structure and its role in microcolony formation. Our results demonstrate that Pel is composed of cationic amino sugars. Using this knowledge, we have developed a Pel-specific lectin stain to directly visualize Pel in biofilms. We show that the positive charge on Pel facilitates its binding to extracellular DNA in the biofilm stalk, and that Pel can compensate for lack of Psl in the biofilm periphery. Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa , Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel’s chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N -acetylgalactosamine and N -acetylglucosamine. Guided by the knowledge of Pel’s sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.

CRISPR-Cas9, which imparts adaptive immunity against foreign genomic invaders in certain prokaryotes, has been repurposed for genome-engineering applications. More recently, another RNA-guided CRISPR endonuclease called Cpf1 (also known as Cas12a) was identified and is also being repurposed. Little is known about the kinetics and mechanism of Cpf1 DNA interaction and how sequence mismatches between the DNA target and guide-RNA influence this interaction. We used single-molecule fluorescence analysis and biochemical assays to characterize DNA interrogation, cleavage, and product release by three Cpf1 orthologs. Our Cpf1 data are consistent with the DNA interrogation mechanism proposed for Cas9. They both bind any DNA in search of protospacer-adjacent motif (PAM) sequences, verify the target sequence directionally from the PAM-proximal end, and rapidly reject any targets that lack a PAM or that are poorly matched with the guide-RNA. Unlike Cas9, which requires 9 bp for stable binding and ∼16 bp for cleavage, Cpf1 requires an ∼17-bp sequence match for both stable binding and cleavage. Unlike Cas9, which does not release the DNA cleavage products, Cpf1 rapidly releases the PAM-distal cleavage product, but not the PAM-proximal product. Solution pH, reducing conditions, and 5′ guanine in guide-RNA differentially affected different Cpf1 orthologs. Our findings have important implications on Cpf1-based genome engineering and manipulation applications.

Structural maintenance of chromosomes (SMC) complexes play critical roles in chromosome dynamics in virtually all organisms, but how they function remains poorly understood. In the bacterium Bacillus subtilis, SMC-condensin complexes are topologically loaded at centromeric sites adjacent to the replication origin. Here we provide evidence that these ring-shaped assemblies tether the left and right chromosome arms together while traveling from the origin to the terminus (>2 megabases) at rates >50 kilobases per minute. Condensin movement scales linearly with time, providing evidence for an active transport mechanism. These data support a model in which SMC complexes function by processively enlarging DNA loops. Loop formation followed by processive enlargement provides a mechanism by which condensin complexes compact and resolve sister chromatids in mitosis and by which cohesin generates topologically associating domains during interphase.

Conventional CRISPR–Cas systems maintain genomic integrity by leveraging guide RNAs for the nuclease-dependent degradation of mobile genetic elements, including plasmids and viruses. Here we describe a notable inversion of this paradigm, in which bacterial Tn 7 -like transposons have co-opted nuclease-deficient CRISPR–Cas systems to catalyse RNA-guided integration of mobile genetic elements into the genome. Programmable transposition of Vibrio cholerae Tn 6677 in Escherichia coli requires CRISPR- and transposon-associated molecular machineries, including a co-complex between the DNA-targeting complex Cascade and the transposition protein TniQ. Integration of donor DNA occurs in one of two possible orientations at a fixed distance downstream of target DNA sequences, and can accommodate variable length genetic payloads. Deep-sequencing experiments reveal highly specific, genome-wide DNA insertion across dozens of unique target sites. This discovery of a fully programmable, RNA-guided integrase lays the foundation for genomic manipulations that obviate the requirements for double-strand breaks and homology-directed repair. A programmable transposase integrates donor DNA at user-defined genomic target sites with high fidelity, revealing a new approach for genetic engineering that obviates the need for DNA double-strand breaks and homologous recombination. 

A detailed understanding of the mechanisms that underlie antibiotic killing is important for the derivation of new classes of antibiotics and clinically useful adjuvants for current antimicrobial therapies. Our efforts to understand why DinB (DNA polymerase IV) overproduction is cytotoxic to Escherichia coli led to the unexpected insight that oxidation of guanine to 8-oxo-guanine in the nucleotide pool underlies much of the cell death caused by both DinB overproduction and bactericidal antibiotics. We propose a model in which the cytotoxicity of beta-lactams and quinolones predominantly results from lethal double-strand DNA breaks caused by incomplete repair of closely spaced 8-oxo-deoxyguanosine lesions, whereas the cytotoxicity of aminoglycosides might additionally result from mistranslation due to the incorporation of 8-oxo-guanine into newly synthesized RNAs.

ADP-ribosyltransferases use NAD + to catalyse substrate ADP-ribosylation 1 , and thereby regulate cellular pathways or contribute to toxin-mediated pathogenicity of bacteria 2 – 4 . Reversible ADP-ribosylation has traditionally been considered a protein-specific modification 5 , but recent in vitro studies have suggested nucleic acids as targets 6 – 9 . Here we present evidence that specific, reversible ADP-ribosylation of DNA on thymidine bases occurs in cellulo through the DarT–DarG toxin–antitoxin system, which is found in a variety of bacteria (including global pathogens such as Mycobacterium tuberculosis , enteropathogenic Escherichia coli and Pseudomonas aeruginosa ) 10 . We report the structure of DarT, which identifies this protein as a diverged member of the PARP family. We provide a set of high-resolution structures of this enzyme in ligand-free and pre- and post-reaction states, which reveals a specialized mechanism of catalysis that includes a key active-site arginine that extends the canonical ADP-ribosyltransferase toolkit. Comparison with PARP–HPF1, a well-established DNA repair protein ADP-ribosylation complex, offers insights into how the DarT class of ADP-ribosyltransferases evolved into specific DNA-modifying enzymes. Together, our structural and mechanistic data provide details of this PARP family member and contribute to a fundamental understanding of the ADP-ribosylation of nucleic acids. We also show that thymine-linked ADP-ribose DNA adducts reversed by DarG antitoxin (functioning as a noncanonical DNA repair factor) are used not only for targeted DNA damage to induce toxicity, but also as a signalling strategy for cellular processes. Using M. tuberculosis as an exemplar, we show that DarT–DarG regulates growth by ADP-ribosylation of DNA at the origin of chromosome replication. Structural and mechanistic data of the ADP-ribosyltransferase DarT demonstrate the role of ADP-ribosylation of DNA by this enzyme in generating toxicity and regulating cellular signalling processes in bacteria.

Efficient and accurate termination is required for gene transcription in all living organisms 1 , 2 . Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway 3 , 4 —called intrinsic termination transcription in bacteria—in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases. Structural studies of Escherichia coli transcription intrinsic termination complexes representing distinct intermediates using cryo-electron microscopy provide insights into the steps and mechanism of transcription termination.

Bacterial chromosomes are folded to compact DNA and facilitate cellular processes. Studying model bacteria has revealed aspects of chromosome folding that are applicable to many species. Primarily controlled by nucleoid-associated proteins, chromosome folding is hierarchical, from large-scale macrodomains to smaller-scale structures that influence DNA transactions, including replication and transcription. Here we review the environmentally regulated, architectural and regulatory roles of nucleoid-associated proteins and the implications for bacterial cell biology. We also highlight similarities and differences in the chromosome folding mechanisms of bacteria and eukaryotes.Advances in sequencing- and imaging-based techniques for chromosome structure analysis have led to a mature understanding of bacterial chromosome structure and dynamics. In this Review, Dame, Rashid and Grainger discuss the hierarchical nature of bacterial chromosome structure and how it is influenced by diverse types of nucleoid-associated proteins. Furthermore, they describe roles for nucleoid-associated proteins and chromosome structure, including in gene expression, chromosome segregation and cell cycle regulation.

Bacterial biofilm infections account for a major proportion of chronic and medical device associated infections in humans, yet our ability to control them is compromised by their inherent tolerance to antimicrobial agents. Cold atmospheric plasma (CAP) represents a promising therapeutic option. CAP treatment of microbial biofilms represents the convergence of two complex phenomena: the production of a chemically diverse mixture of reactive species and intermediates, and their interaction with a heterogeneous 3D interface created by the biofilm extracellular polymeric matrix. Therefore, understanding these interactions and physiological responses to CAP exposure are central to effective management of infectious biofilms. We review the unique opportunities and challenges for translating CAP to the management of biofilms. Biofilms are implicated in around 65% of all chronic human infections, including those associated with indwelling medical devices such as catheters and prostheses. Biofilm infections are often asymptomatic between exacerbations and challenging to detect and effectively treat using conventional antibiotics and antimicrobial agents. CAP provides an effective multimodal, multitarget approach for controlling microbial biofilms. Biofilms express a complex extracellular matrix of polymeric substances that may attenuate the antimicrobial efficacy of CAP via interactions with CAP-generated RONS. Biofilm tolerance to CAP is variable between species and between strains of the same species, which may be due to production of EPS, RONS-detoxifying enzymes, or acquired tolerance to physiological RONS during chronic infections.