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.
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, σ 70 , 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.
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.
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.
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.
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.
EcoCyc is a bioinformatics database (DB) available at EcoCyc.org that describes the genome and the biochemical machinery of Escherichia coli K-12 MG1655. The long-term goal of the project was to describe the complete molecular catalog of the E. coli cell, as well as the functions of each of its molecular parts, to facilitate a system-level understanding of E. coli . EcoCyc is an electronic reference source for E. coli biologists and for biologists who work with related microorganisms. The database includes information pages on each E. coli gene product, metabolite, reaction, operon, and metabolic pathway. The database also includes information on the regulation of gene expression, E. coli gene essentiality, and nutrient conditions that do or do not support the growth of E. coli . The website and downloadable software contain tools for the analysis of high-throughput data sets. In addition, a steady-state metabolic flux model is generated from each new version of EcoCyc and can be executed via EcoCyc.org. The model can predict metabolic flux rates, nutrient uptake rates, and growth rates for different gene knockouts and nutrient conditions. Data generated from a whole-cell model that is parameterized from the latest data on EcoCyc is also available. This review outlines the data content of EcoCyc and the procedures by which this content is generated.
The discovery and subsequent characterization and applications of CRISPR-Cas is one of the most fascinating scientific stories from the past two decades. While first identified in Escherichia coli , this microbial workhorse often took a back seat to other bacteria during the early race to detail CRISPR-Cas function as an adaptive immune system. This was not a deliberate slight, but the result of early observations that the CRISPR-Cas systems found in E. coli were not robust phage defense systems as first described in Streptococcus thermophilus . This apparent lack of activity was discovered to result from transcriptional repression by the nucleoid protein H-NS. Despite extensive evidence arguing against such roles, some studies still present E. coli CRISPR-Cas systems in the context of anti-phage and/or anti-plasmid activities. Here, the studies that led to our understanding of its cryptic nature are highlighted, along with ongoing research to uncover potential alternative functions in E. coli .
Siderophore cephalosporins are designed to exploit bacterial nutrient uptake systems to gain accelerated uptake across the outer membrane of Gram-negative bacteria. They contain iron (III) binding motifs that allow them to form complexes that will be recognized as potential substrates by iron-siderophore transport systems. Research during the last five decades has culminated in the approval for clinical use of the siderophore cephalosporin cefiderocol, which incorporates accumulated learning from investigations of structural features that enhance resistance toward hydrolysis by β-lactamases, that promote bacterial membrane permeability, and that confer long pharmacokinetic half-life in the human host.
ABSTRACT 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.
Salmonella enterica is a diverse species that infects both humans and animals. S. enterica subspecies enterica consists of more than 1,500 serovars. Unlike typhoidal Salmonella serovars which are human host-restricted, non-typhoidal Salmonella (NTS) serovars are associated with foodborne illnesses worldwide and are transmitted via the food chain. Additionally, NTS serovars can cause disease in livestock animals causing significant economic losses. Salmonella is a well-studied model organism that is easy to manipulate and evaluate in animal models of infection. Advances in genetic engineering approaches in recent years have led to the development of Salmonella vaccines for both humans and animals. In this review, we focus on current progress of recombinant live-attenuated Salmonella vaccines, their use as a source of antigens for parenteral vaccines, their use as live-vector vaccines to deliver foreign antigens, and their use as therapeutic cancer vaccines in humans. We also describe development of live-attenuated Salmonella vaccines and live-vector vaccines for use in animals.
Escherichia coli has been a vital model organism for studying chromosomal structure, thanks, in part, to its small and circular genome (4.6 million base pairs) and well-characterized biochemical pathways. Over the last several decades, we have made considerable progress in understanding the intricacies of the structure and subsequent function of the E. coli nucleoid. At the smallest scale, DNA, with no physical constraints, takes on a shape reminiscent of a randomly twisted cable, forming mostly random coils but partly affected by its stiffness. This ball-of-spaghetti-like shape forms a structure several times too large to fit into the cell. Once the physiological constraints of the cell are added, the DNA takes on overtwisted (negatively supercoiled) structures, which are shaped by an intricate interplay of many proteins carrying out essential biological processes. At shorter length scales (up to about 1 kb), nucleoid-associated proteins organize and condense the chromosome by inducing loops, bends, and forming bridges. Zooming out further and including cellular processes, topological domains are formed, which are flanked by supercoiling barriers. At the megabase-scale both large, highly self-interacting regions (macrodomains) and strong contacts between distant but co-regulated genes have been observed. At the largest scale, the nucleoid forms a helical ellipsoid. In this review, we will explore the history and recent advances that pave the way for a better understanding of E. coli chromosome organization and structure, discussing the cellular processes that drive changes in DNA shape, and what contributes to compaction and formation of dynamic structures, and in turn how bacterial chromatin affects key processes such as transcription and replication.
ABSTRACT In this chapter, we update our 2004 review of “The Life of Commensal Escherichia coli in the Mammalian Intestine” ( https://doi.org/10.1128/ecosalplus.8.3.1.2 ), with a change of title that reflects the current focus on “Nutrition of E. coli within the Intestinal Microbiome.” The earlier part of the previous two decades saw incremental improvements in understanding the carbon and energy sources that E. coli and Salmonella use to support intestinal colonization. Along with these investigations of electron donors came a better understanding of the electron acceptors that support the respiration of these facultative anaerobes in the gastrointestinal tract. Hundreds of recent papers add to what was known about the nutrition of commensal and pathogenic enteric bacteria. The fact that each biotype or pathotype grows on a different subset of the available nutrients suggested a mechanism for succession of commensal colonizers and invasion by enteric pathogens. Competition for nutrients in the intestine has also come to be recognized as one basis for colonization resistance, in which colonized strain(s) prevent colonization by a challenger. In the past decade, detailed investigations of fiber- and mucin-degrading anaerobes added greatly to our understanding of how complex polysaccharides support the hundreds of intestinal microbiome species. It is now clear that facultative anaerobes, which usually cannot degrade complex polysaccharides, live in symbiosis with the anaerobic degraders. This concept led to the “restaurant hypothesis,” which emphasizes that facultative bacteria, such as E. coli , colonize the intestine as members of mixed biofilms and obtain the sugars they need for growth locally through cross-feeding from polysaccharide-degrading anaerobes. Each restaurant represents an intestinal niche. Competition for those niches determines whether or not invaders are able to overcome colonization resistance and become established. Topics centered on the nutritional basis of intestinal colonization and gastrointestinal health are explored here in detail.
ABSTRACT Toxin-antitoxin systems are ubiquitous in the prokaryotic world and widely distributed among chromosomes and mobile genetic elements. Several different toxin-antitoxin system types exist, but what they all have in common is that toxin activity is prevented by the cognate antitoxin. In type I toxin-antitoxin systems, toxin production is controlled by an RNA antitoxin and by structural features inherent to the toxin messenger RNA. Most type I toxins are small membrane proteins that display a variety of cellular effects. While originally discovered as modules that stabilize plasmids, chromosomal type I toxin-antitoxin systems may also stabilize prophages, or serve important functions upon certain stress conditions and contribute to population-wide survival strategies. Here, we will describe the intricate RNA-based regulation of type I toxin-antitoxin systems and discuss their potential biological functions.
Salmonella enterica is the leading cause of bacterial foodborne illness in the USA, with an estimated 95% of salmonellosis cases due to the consumption of contaminated food products. Salmonella can cause several different disease syndromes, with the most common being gastroenteritis, followed by bacteremia and typhoid fever. Among the over 2,600 currently identified serotypes/serovars, some are mostly host-restricted and host-adapted, while the majority of serotypes can infect a broader range of host species and are associated with causing both livestock and human disease. Salmonella serotypes and strains within serovars can vary considerably in the severity of disease that may result from infection, with some serovars that are more highly associated with invasive disease in humans, while others predominantly cause mild gastroenteritis. These observed clinical differences may be caused by the genetic make-up and diversity of the serovars. Salmonella virulence systems are very complex containing several virulence-associated genes with different functions that contribute to its pathogenicity. The different clinical syndromes are associated with unique groups of virulence genes, and strains often differ in the array of virulence traits they display. On the chromosome, virulence genes are often clustered in regions known as Salmonella pathogenicity islands (SPIs), which are scattered throughout different Salmonella genomes and encode factors essential for adhesion, invasion, survival, and replication within the host. Plasmids can also carry various genes that contribute to Salmonella pathogenicity. For example, strains from several serovars associated with significant human disease, including Choleraesuis, Dublin, Enteritidis, Newport, and Typhimurium, can carry virulence plasmids with genes contributing to attachment, immune system evasion, and other roles. The goal of this comprehensive review is to provide key information on the Salmonella virulence, including the contributions of genes encoded in SPIs and plasmids during Salmonella pathogenesis.
Promoter-specific activation of transcript initiation provides an important regulatory device in Escherichia coli and Salmonella. Here, we describe the different mechanisms that operate, focusing on how they have evolved to manage the "housekeeping" bacterial transcription machinery. Some mechanisms involve assisting the bacterial DNA-dependent RNA polymerase or replacing or remodeling one of its subunits. Others are directed to chromosomal DNA, improving promoter function, or relieving repression. We discuss how different activators work together at promoters and how the present complex network of transcription factors evolved.
