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.

EcoSal Plus (ESP) is the authoritative online review journal that publishes an ever-growing body of expert reviews covering virtually all aspects of Escherichia coli, Salmonella, and other members of the order Enterobacterales and their use as model microbes for biological explorations. This review will cover the history of ESP, starting with its origins as multi-volume printed books entitled Escherichia coli and Salmonella: Cellular and Molecular Biology that became "the Bible" for information on the physiology, metabolism, genetics, and other aspects of E. coli and Salmonella. After two printed editions, this resource moved online as EcoSal in an era when electronic publishing was still in its infancy. Progress in establishing EcoSal was slow due to technical issues of online publishing and difficulties in recruiting authors to produce new material. This venture was relaunched in 2013 as EcoSal Plus in a completely new web platform that was much more user (and author) friendly and with an expanded scope to include other members of the order Enterobacterales. EcoSal Plus will be ending as a standalone publication but will merge with Microbiology and Molecular Biology Reviews to continue providing high-quality, authoritative reviews on E. coli, Salmonella, and related organisms.

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.

Immediately after infection of Escherichia coli, bacteriophage T4 begins to reprogram the host's transcriptional machinery, first by chemical modification and then by producing factors that alter the specificity of RNA polymerase (RNAP). This leads to the temporal expression of three classes of T4 transcripts: early, middle, and late. For early transcription, the Alt protein, which is present in the phage head, is injected with the DNA and subsequently ADP-ribosylates RNAP, providing an advantage for T4 early promoters over host promoters. For middle and late transcription, T4 utilizes phage-encoded factors to either reconfigure or replace the primary specificity subunit, σ70, of RNAP, respectively. In both cases, the phage relies on several processes to maximize the efficiency of these phage-created, alternative σ's. This review summarizes older biochemical, genetic, and structural work that elucidated many of the elegant mechanisms of this transcriptional takeover and focuses on the more recent cryo-EM structures of the complete transcription machines that allow us to visualize the processes.

The bacteriophage T4 replisome is a complex molecular machine responsible for DNA replication in the T4 phage. It consists of multiple proteins that work together to ensure efficient and accurate replication of the phage genome. The replisome comprises DNA polymerases, helicase, primase, and other accessory proteins, which coordinate leading- and lagging-strand synthesis. Extensive research over the years, including protein analysis, enzyme kinetics, and structural investigations, has provided insights into the organization and function of these proteins, along with their dynamics and coordination at the replication fork. The T4 replisome serves as a useful model system for understanding molecular fidelity, enzymatic interplay, and fundamental principles of DNA replication.

Understanding the mechanisms that modulate horizontal genetic exchange in prokaryotes is a key problem in biology. DNA entry is limited by resident host-dependent restriction-modification (RM) systems (HDRM), which are present in most prokaryotic genomes. This review specifically focuses on the biological functions of HDRM, rather than detailed enzyme mechanisms. DNA in each cell carries epigenetic marks imposed by host-modifying enzymes (HDM), most often not only base methylation but also additions to the phosphodiester backbone. The pattern of base and backbone modifications is read by host-restriction enzymes (HDR). Broadly, HDRM systems read the pattern of chemical modifications to DNA at host-determined (HD) sites to regulate the fate of incoming mobile DNA. An inappropriate pattern may be restricted either due to the absence of protective modification or its presence; the latter activity is mediated by modification-dependent restriction enzymes (MDRE). Most often, restriction occurs via nuclease-mediated degradation, but it can also act via other mechanisms that prevent the initiation of replication. Like other genome-defense systems, HDRM systems are highly diverse and somewhat modular. The basic functions required for action in vivo and the protein domains responsible for each function are addressed here. Particularly under-studied among the latter are the interaction domains that control the launch of highly toxic activities such as HDR. These have been evolutionarily shuffled to build a variety of classical RM systems as well as more divergent systems.

Bacteriophage T4 has provided a model system for understanding post-transcriptional regulation in prokaryotes. This review summarizes several mechanisms of RNA processing and translational control in T4 infection, focusing on the coordinated actions of phage and host RNases. Key regulators such as RNase E, RegB, and tRNA-processing enzymes are discussed, along with the roles of RNA secondary structures and the translational repressors gp32, gp43, and RegA. In addition, we review recent studies that show how the host's antiphage toxin-antitoxin defense systems target T4 mRNAs as well as counter-strategies by the phage. Together, these components help to ensure temporal precision and efficiency of phage gene expression during phage infection.

The chromosomal DNA of Escherichia coli is approximately a thousand times longer than the linear dimensions of the cell it occupies. Nevertheless, it fills only about one-half of the cytosolic volume of the cell. The volume pervaded by the chromosomal DNA is known as nucleoid. The nucleoid is a ribosome-depleted region that behaves as a distinct liquid-like phase within the cytosol. In most bacteria, including E. coli, which lack membrane-enclosed organelles, the phase separation between the nucleoid and the ribosome-rich cytosolic fraction represents the most prominent organizational principle of the cell's cytosolic interior. This review explores the mechanisms driving nucleoid phase separation, including the roles of DNA-binding proteins, supercoiling, and active DNA looping. Recent studies highlight macromolecular crowding as the dominant factor governing this spatial organization. The main focus of this review is on experimental and theoretical works-ranging from in vitro and in vivo studies to polymer physics-based models-that elucidate how macromolecular crowding drives nucleoid phase formation and regulates DNA compaction in E. coli.

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.

A cause of diarrhea worldwide, enteroaggregative Escherichia coli (or EAEC) is one of six diarrheagenic E. coli pathotypes. EAEC strains are heterogeneic in terms of virulence factors, adhere strongly to epithelial cells, and produce a strong biofilm. It is the characteristic aggregative adherence on epithelial cells that was both the gold standard of clinical identification and the source of the appellation "aggregative." To understand EAEC in the continuum with other pathogenic E. coli, we discuss the overlap of EAEC with other diarrheagenic E. coli and extraintestinal pathogenic E. coli isolates. Due to the increased use of molecular techniques for the identification of EAEC, the use of various PCR markers and DNA sequencing for EAEC identification and how that correlates to the phenotypic definition is discussed. Aspects of EAEC pathogenesis, including an overview of virulence factors, such as the five aggregative adherence fimbriae (AAF) and SPATEs (serine protease autotransporters of Enterobacteriaceae), will be explored. The advantages and limitations of various EAEC animal models and what is known about human immunity and host factors that influence infection outcomes are outlined. This review includes a synthesis of new discoveries published for the EAEC field, including non-AAF fimbrial adhesins, additional information about post-infection sequelae, and new EAEC models.