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Listeria monocytogenes is a food-borne pathogen responsible for a disease called listeriosis, which is potentially lethal in immunocompromised individuals. This bacterium, first used as a model to study cell-mediated immunity, has emerged over the past 20 years as a paradigm in infection biology, cell biology and fundamental microbiology. In this Review, we highlight recent advances in the understanding of human listeriosis and L. monocytogenes biology. We describe unsuspected modes of hijacking host cell biology, ranging from changes in organelle morphology to direct effects on host transcription via a new class of bacterial effectors called nucleomodulins. We then discuss advances in understanding infection in vivo, including the discovery of tissue-specific virulence factors and the 'arms race' among bacteria competing for a niche in the microbiota. Finally, we describe the complexity of bacterial regulation and physiology, incorporating new insights into the mechanisms of action of a series of riboregulators that are critical for efficient metabolic regulation, antibiotic resistance and interspecies competition.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M.tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting.

Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA 1 (msDNA). Despite decades of research on the biosynthesis of msDNA 2 , the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella enterica serovar Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase–msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a new type of tripartite DNA-containing toxin–antitoxin system. To understand the physiological roles of such toxin–antitoxin systems, we developed toxin activation–inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin–antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin–antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports 3 , 4 . Thus, RcaT retrons are tripartite DNA-regulated toxin–antitoxin systems, which use the reverse transcriptase–msDNA complex both as an antitoxin and as a sensor of phage protein activities. Retron-Sen2 of Salmonella Typhimurium encodes a toxin and a reverse transcriptase, which, together with the Sen2 multi-copy single-stranded DNA synthesized by the reverse transcriptase make up a tripartite toxin–antitoxin system that functions in anti-phage defence.

Prokaryotic organisms are threatened by a large array of viruses and have developed numerous defence strategies. Among these, only clustered, regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity against foreign elements. Upon viral injection, a small sequence of the viral genome, known as a spacer, is integrated into the CRISPR locus to immunize the host cell. Spacers are transcribed into small RNA guides that direct the cleavage of the viral DNA by Cas nucleases. Immunization through spacer acquisition enables a unique form of evolution whereby a population not only rapidly acquires resistance to its predators but also passes this resistance mechanism vertically to its progeny. The CRISPR-Cas systems of bacteria and archaea provide adaptive immunity against invading mobile genetic elements such as phages and plasmids; this Review describes the discovery of these systems and the mechanisms of immunity, including recent progress in establishing the molecular basis of host immunization. CRISPR–Cas immunity system dissected The CRISPR–Cas systems of bacteria and archaea provide adaptive immunity against invading mobile genetic elements (MGEs), such as phages and plasmids. These systems function through the use of small guide RNAs derived from previous encounters with MGEs, which together with the Cas proteins, function in the degradation of complementary invading nucleic acids. In this Review, Luciano Marraffini describes the discovery of these systems and the mechanisms of immunity, including recent progress in establishing the molecular basis of host immunization. The author also describes the main outstanding questions in the field and the directions for future progress.

In all domains of life, genomes contain epigenetic information superimposed over the nucleotide sequence. Epigenetic signals control DNA–protein interactions and can cause phenotypic change in the absence of mutation. A nearly universal mechanism of epigenetic signalling is DNA methylation. In bacteria, DNA methylation has roles in genome defence, chromosome replication and segregation, nucleoid organization, cell cycle control, DNA repair and regulation of transcription. In many bacterial species, DNA methylation controls reversible switching (phase variation) of gene expression, a phenomenon that generates phenotypic cell variants. The formation of epigenetic lineages enables the adaptation of bacterial populations to harsh or changing environments and modulates the interaction of pathogens with their eukaryotic hosts.

Key Points This Review discusses natural bacterial transformation, highlighting the common and divergent features that exist in a phylogenetically diverse range of naturally transformable species. Transformation is defined as the uptake of foreign DNA as single strands and its subsequent integration into the bacterial chromosome by homologous recombination. The mechanisms of uptake and integration, which are largely conserved among species, are highlighted and their conservation is explored. In contrast to DNA-uptake mechanisms, the regulation of the ability to transform (which is known as competence) and the signals that induce competence vary widely between species; the range of mechanisms that are involved are discussed. The roles of competence and imported DNA are also considered, and we argue that evidence so far generally points towards a role for transformation in the generation of genetic diversity or in chromosomal repair, rather than a nutritional role. Finally, we explore the future prospects in this field of research, detailing several case studies of species that have recently been shown to be transformable and the potential difficulties in demonstrating transformability in a new species. In this Review, Claverys and colleagues describe the divergent and common principles that govern the transformation process in phylogenetically distinct bacteria and discuss the potential role of imported DNA in generating genetic diversity. They also discuss how this information can be used for the prediction of new transformable species. Natural bacterial transformation involves the internalization and chromosomal integration of DNA and has now been documented in ∼80 species. Recent advances have established that phylogenetically distant species share conserved uptake and processing proteins but differ in the inducing cues and regulatory mechanisms that are involved. In this Review, we highlight divergent and common principles that govern the transformation process in different bacteria. We discuss how this cumulative knowledge enables the prediction of new transformable species and supports the idea that the main role of internalized DNA is in the generation of genetic diversity or in chromosome repair rather than in nutrition.

Bacteria are continuously exposed to numerous endogenous and exogenous DNA-damaging agents. To maintain genome integrity and ensure cell survival, bacteria have evolved several DNA repair pathways to correct different types of DNA damage and non-canonical bases, including strand breaks, nucleotide modifications, cross-links, mismatches and ribonucleotide incorporations. Recent advances in genome-wide screens, the availability of thousands of whole-genome sequences and advances in structural biology have enabled the rapid discovery and characterization of novel bacterial DNA repair pathways and new enzymatic activities. In this Review, we discuss recent advances in our understanding of base excision repair and nucleotide excision repair, and we discuss several new repair processes including the EndoMS mismatch correction pathway and the MrfAB excision repair system.To maintain genome integrity and ensure cell survival, bacteria have evolved several DNA repair pathways to repair different types of DNA damage and non-canonical bases, including strand breaks, nucleotide modifications, cross-links, mismatches and ribonucleotide incorporations. In this Review, Wozniak and Simmons provide a contemporary discussion of the excision-based mechanisms bacteria use to repair the diverse set of lesions they encounter.

Five classes of phage genes are identified that protect phages from CRISPR-mediated bacterial immunity. Phage genes deploy 'anti-CRISPR' defence CRISPR/Cas immune systems, widely distributed in bacteria and Archaea, protect microbial cells from phage attack through the use of small RNAs for sequence-specific detection and neutralization of invading genomes. It has been suggested that 'anti-CRISPR' mechanisms might exist, and here Alan Davidson and colleagues identify phage-encoded factors that inhibit the CRISPR/Cas system. They also find homologues of these genes in Pseudomonas species, indicating that anti-CRISPR elements have a critical role in the evolution of this bacterial pathogen. A widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules consists of the clustered regularly interspaced short palindromic repeats (CRISPR) coupled with cas (CRISPR-associated) genes 1 . Similar to RNA interference in eukaryotes 2 , these CRISPR/Cas systems use small RNAs for sequence-specific detection and neutralization of invading genomes 3 . Here we describe the first examples of genes that mediate the inhibition of a CRISPR/Cas system. Five distinct ‘anti-CRISPR’ genes were found in the genomes of bacteriophages infecting Pseudomonas aeruginosa . Mutation of the anti-CRISPR gene of a phage rendered it unable to infect bacteria with a functional CRISPR/Cas system, and the addition of the same gene to the genome of a CRISPR/Cas-targeted phage allowed it to evade the CRISPR/Cas system. Phage-encoded anti-CRISPR genes may represent a widespread mechanism for phages to overcome the highly prevalent CRISPR/Cas systems. The existence of anti-CRISPR genes presents new avenues for the elucidation of CRISPR/Cas functional mechanisms and provides new insight into the co-evolution of phages and bacteria.

Argonaute proteins constitute a highly diverse family of nucleic acid-guided proteins. They were first discovered in eukaryotes as key proteins in RNA interference systems, but homologous prokaryotic Argonaute proteins (pAgos) have also been found in archaea and bacteria. In this Progress article, we focus on long pAgo variants, a class of pAgos that are involved in nucleic acid-guided host defence against invading nucleic acids, and discuss the potential of pAgos in genome editing.

In this Opinion article, Herskovits and colleagues introduce an emerging class of bacteria–phage symbiotic interaction — which they term 'active lysogeny' — in which phages regulate the expression of bacterial genes by precise insertion and excision events. Unlike lytic phages, temperate phages that enter lysogeny maintain a long-term association with their bacterial host. In this context, mutually beneficial interactions can evolve that support efficient reproduction of both phages and bacteria. Temperate phages are integrated into the bacterial chromosome as large DNA insertions that can disrupt gene expression, and they may pose a fitness burden on the cell. However, they have also been shown to benefit their bacterial hosts by providing new functions in a bacterium–phage symbiotic interaction termed lysogenic conversion. In this Opinion article, we discuss another type of bacterium–phage interaction, active lysogeny, in which phages or phage-like elements are integrated into the bacterial chromosome within critical genes or operons and serve as switches that regulate bacterial genes via genome excision.