The homologous recombination (HR) system of bacteriophage T4 plays critical, direct roles in the replication and repair of the phage genome. This review covers the classic, UvsX-dependent HR pathway in T4, focusing on recent findings on the mechanisms of central HR proteins UvsX, UvsY, and Gp32, plus the key helicase and nuclease enzymes that affect HR and promote its coupling to T4 recombination-dependent replication and repair processes. The T4 HR pathways are paradigmatic, since they are highly conserved in all orders of viral and cellular life. Therefore, the study of T4 recombination is highly relevant to biomedicine and to environmental microbiology. At the same time, the tractability of the T4 recombination system for biochemical studies has led to the development of novel, isothermal DNA amplification technologies based on the activities of UvsX, UvsY, and Gp32, which are discussed herein. Globally, the recent revolution in metagenomics has demonstrated that T4-like phages, most encoding the genes and proteins of the T4 HR system, are abundant and widespread in the environment, where they play important roles in the dynamics of diverse microbiomes, from the earth's oceans to the animal gut. Accordingly, we discuss the conservation of T4 HR genes in representatives of T4-like jumbo phages and cyanophages. As a paradigm for HR in diverse organisms, as a source of novel technologies, and as a window on the importance of bacteriophages in the environment, the T4 HR system continues to provide new insights and reagents for a better understanding of life on earth.

It has been a 45-year journey studying genome packaging of a single virus, the tailed bacteriophage T4. T4, then and now, remains a powerful model for understanding viruses, particularly tailed phages, the most abundant and widely distributed organisms on Earth. The biochemistry, structure, and single-molecule dynamics of the T4 DNA packaging motor have been teased out. Packaging ~171 kb genomic DNA into a 120 × 86 nm prolate icosahedral head in a few minutes, the T4 packaging motor is the fastest and most powerful motor known. It is also the most promiscuous, allowing packaging of any double-stranded DNA regardless of sequence or length into various head (capsid) assemblies: unexpanded prohead, expanded prohead, or mature head. These studies established the basic architecture of an ATP-powered viral genome packaging machine consisting of a pentameric packaging motor attached to the dodecameric portal vertex of the capsid shell. Furthermore, it opened new avenues to engineer and repurpose the packaging machine for the delivery of genes, proteins, and protein-nucleic acid complexes into human cells. The biggest challenge now is to translate this knowledge into the design of future phage-vectored gene therapy platforms that allow engineered phages to interact with human cells and make appropriate genetic and metabolic corrections to alleviate disease. This possibility was unimaginable when we started but evolved through lessons learned by examining the intricate machinery of the phage T4 life cycle.

Escherichia coli and non-typhoidal Salmonella enterica are capable of persisting and growing in a wide range of environments. Although best known for their interactions and pathogenic phenotypes in warm-blooded animal hosts, they can be located in a diversity of hosts and habitats. This capability has led to foodborne illness arising from multiple sources, including crop plants. It raises key questions about the bacterial traits and adaptations that permit this degree of flexibility. By describing plant features and the associated environments, we illustrate the underlying physiological basis that enables E. coli, including Shiga toxin-producing E. coli, and S. enterica to colonize plant hosts. We follow the distinct stages of the interactions and the different considerations to understand how they will play out and the resulting outcome for the bacteria. Knowledge of the processes involved lays the foundation for understanding and managing real-life scenarios in agriculture and food production and allows predictions for the bacterial responses in the plant environment under changing climatic conditions.

In the face of rising antimicrobial resistance, bacteriophage therapy, also known as phage therapy, is seeing a resurgence as a potential treatment for bacterial infections including urinary tract infection (UTI). Primarily caused by uropathogenic Escherichia coli, the 400 million UTI cases annually are major global healthcare burdens and a primary cause of antibiotic prescriptions in the outpatient setting. Phage therapy has several potential advantages over antibiotics including the ability to disrupt bacterial biofilms and synergize with antimicrobial treatments with minimal side effects or impacts on the microbiota. Phage therapy for UTI treatment has shown generally favorable results in recent animal models and human case reports. Ongoing clinical trials seek to understand the efficacy of phage therapy in individuals with asymptomatic bacteriuria and uncomplicated cystitis. A possible challenge for phage therapy is the development of phage resistance in bacteria during treatment. While resistance frequently develops in vitro and in vivo, resistance can come with negative consequences for the bacteria, leaving them susceptible to antibiotics and other environmental conditions and reducing their overall virulence. "Steering" bacteria toward phage resistance outcomes that leave them less fit or virulent is especially useful in the context of UTI where poorly adherent or slow-growing bacteria are likely to be flushed from the system. In this article, we describe the history of phage therapy in treating UTI and its current resurgence, the state of its clinical use, and an outlook on how well-designed phage therapy could be used to "steer" bacteria toward less virulent and antimicrobial-susceptible states.

Environments inhabited by Enterobacteriaceae are diverse and often stressful. This is particularly true for Escherichia coli and Salmonella during host association in the gastrointestinal systems of animals. There, E. coli and Salmonella must survive exposure to various antimicrobial compounds produced or ingested by their host. A myriad of changes to cellular physiology and metabolism are required to achieve this feat. A central regulatory network responsible for sensing and responding to intracellular chemical stressors like antibiotics are the Mar, Sox, and Rob systems found throughout the Enterobacteriaceae. Each of these distinct regulatory networks controls expression of an overlapping set of downstream genes whose collective effects result in increased resistance to a wide array of antimicrobial compounds. This collection of genes is known as the mar-sox-rob regulon. This review will provide an overview of the mar-sox-rob regulon and molecular architecture of the Mar, Sox, and Rob systems.

Antibiotic resistance is a major public health threat that has stimulated the scientific community to search for nontraditional therapeutic targets. Because virulence, but not the growth, of many Gram-negative bacterial pathogens depends on the multicomponent type three secretion system injectisome (T3SSi), the T3SSi has been an attractive target for identifying small molecules, peptides, and monoclonal antibodies that inhibit its function to render the pathogen avirulent. While many small-molecule lead compounds have been identified in whole-cell-based high-throughput screens (HTSs), only a few protein targets of these compounds are known; such knowledge is an important step to developing more potent and specific inhibitors. Evaluation of the efficacy of compounds in animal studies is ongoing. Some efforts involving the development of antibodies and vaccines that target the T3SSi are further along and include an antibody that is currently in phase II clinical trials. Continued research into these antivirulence therapies, used alone or in combination with traditional antibiotics, requires combined efforts from both pharmaceutical companies and academic labs.

Gram-negative bacteria have evolved a complex envelope to adapt and survive in a broad range of ecological niches. This physical barrier is the first line of defense against noxious compounds and viral particles called bacteriophages. Colicins are a family of bactericidal proteins produced by and toxic to Escherichia coli and closely related bacteria. Filamentous phages have a complex structure, composed of at least five capsid proteins assembled in a long thread-shaped particle, that protects the viral DNA. Despite their difference in size and complexity, group A colicins and filamentous phages both parasitize multiprotein complexes of their sensitive host for entry. They first bind to a receptor located at the surface of the target bacteria before specifically recruiting components of the Tol system to cross the outer membrane and find their way through the periplasm. The Tol system is thought to use the proton motive force of the inner membrane to maintain outer membrane integrity during the life cycle of the cell. This review describes the sequential docking mechanisms of group A colicins and filamentous phages during their uptake by their bacterial host, with a specific focus on the translocation step, promoted by interactions with the Tol system.

Plasmids are ubiquitous in the microbial world and have been identified in almost all species of bacteria that have been examined. Their localization inside the bacterial cell has been examined for about two decades; typically, they are not randomly distributed, and their positioning depends on copy number and their mode of segregation. Low-copy-number plasmids promote their own stable inheritance in their bacterial hosts by encoding active partition systems, which ensure that copies are positioned in both halves of a dividing cell. High-copy plasmids rely on passive diffusion of some copies, but many remain clustered together in the nucleoid-free regions of the cell. Here we review plasmid localization and partition (Par) systems, with particular emphasis on plasmids from Enterobacteriaceae and on recent results describing the in vivo localization properties and molecular mechanisms of each system. Partition systems also cause plasmid incompatibility such that distinct plasmids (with different replicons) with the same Par system cannot be stably maintained in the same cells. We discuss how partition-mediated incompatibility is a consequence of the partition mechanism.

Type V, or "autotransporter," secretion is a term used to refer to several simple protein export pathways that are found in a wide range of Gram-negative bacteria. Autotransporters are generally single polypeptides that consist of an extracellular ("passenger") domain and a β barrel domain that anchors the protein to the outer membrane (OM). Although it was originally proposed that the passenger domain is secreted through a channel formed solely by the covalently linked β barrel domain, experiments performed primarily on the type Va, or "classical," autotransporter pathway have challenged this hypothesis. Several lines of evidence strongly suggest that both the secretion of the passenger domain and the membrane integration of the β barrel domain are catalyzed by the barrel assembly machinery (Bam) complex, a conserved hetero-oligomer that plays an essential role in the assembly of most integral OM proteins. The secretion reaction appears to be driven at least in part by the folding of the passenger domain in the extracellular space. Although many aspects of autotransporter biogenesis remain to be elucidated, it will be especially interesting to determine whether the different classes of proteins that fall under the type V rubric-most of which have not been examined in detail-are assembled by the same basic mechanism as classical autotransporters.

Gram-negative bacteria assemble a variety of surface structures, including the hair-like organelles known as pili or fimbriae. Pili typically function in adhesion and mediate interactions with various surfaces, with other bacteria, and with other types of cells such as host cells. The chaperone/usher (CU) pathway assembles a widespread class of adhesive and virulence-associated pili. Pilus biogenesis by the CU pathway requires a dedicated periplasmic chaperone and integral outer membrane protein termed the usher, which forms a multifunctional assembly and secretion platform. This review addresses the molecular and biochemical aspects of the CU pathway in detail, focusing on the type 1 and P pili expressed by uropathogenic Escherichia coli as model systems. We provide an overview of representative CU pili expressed by E. coli and Salmonella, and conclude with a discussion of potential approaches to develop antivirulence therapeutics that interfere with pilus assembly or function.