EcoSal Plus最新文献

Over the last decade, the study of CRISPR-Cas systems has progressed from a newly discovered bacterial defense mechanism to a diverse suite of genetic tools that have been applied across all domains of life. While the initial applications of CRISPR-Cas technology fulfilled a need to more precisely edit eukaryotic genomes, creative "repurposing" of this adaptive immune system has led to new approaches for genetic analysis of microorganisms, including improved gene editing, conditional gene regulation, plasmid curing and manipulation, and other novel uses. The main objective of this review is to describe the development and current state-of-the-art use of CRISPR-Cas techniques specifically as it is applied to members of the Enterobacteriales. While many of the applications covered have been initially developed in Escherichia coli, we also highlight the potential, along with the limitations, of this technology for expanding the availability of genetic tools in less-well-characterized non-model species, including bacterial pathogens.

For decades, biologist have exploited the near boundless advantages that molecular and genetic tools and analysis provide for our ability to understand biological systems. One of these genetic tools, suppressor analysis, has proven invaluable in furthering our understanding of biological processes and pathways and in discovering unknown interactions between genes and gene products. The power of suppressor analysis lies in its ability to discover genetic interactions in an unbiased manner, often leading to surprising discoveries. With advancements in technology, high-throughput approaches have aided in large-scale identification of suppressors and have helped provide insight into the core functional mechanisms through which suppressors act. In this review, we examine some of the fundamental discoveries that have been made possible through analysis of suppressor mutations. In addition, we cover the different types of suppressor mutants that can be isolated and the biological insights afforded by each type. Moreover, we provide considerations for the design of experiments to isolate suppressor mutants and for strategies to identify intergenic suppressor mutations. Finally, we provide guidance and example protocols for the isolation and mapping of suppressor mutants.

Escherichia coli and Salmonella isolates produce a range of different polysaccharide structures that play important roles in their biology. E. coli isolates often possess capsular polysaccharides (K antigens), which form a surface structural layer. These possess a wide range of repeat-unit structures. In contrast, only one capsular polymer (Vi antigen) is found in Salmonella, and it is confined to typhoidal serovars. In both genera, capsules are vital virulence determinants and are associated with the avoidance of host immune defenses. Some isolates of these species also produce a largely secreted exopolysaccharide called colanic acid as part of their complex Rcs-regulated phenotypes, but the precise function of this polysaccharide in microbial cell biology is not fully understood. E. coli isolates produce two additional secreted polysaccharides, bacterial cellulose and poly-N-acetylglucosamine, which play important roles in biofilm formation. Cellulose is also produced by Salmonella isolates, but the genes for poly-N-acetylglucosamine synthesis appear to have been lost during its evolution toward enhanced virulence. Here, we discuss the structures, functions, relationships, and sophisticated assembly mechanisms for these important biopolymers.

Yersinia pseudotuberculosis is an Enterobacteriaceae family member that is commonly transmitted by the fecal-oral route to cause infections. From the small intestine, Y. pseudotuberculosis can invade through Peyer's patches and lymph vessels to infect the mesenteric lymph nodes (MLNs). Infection of MLNs by Y. pseudotuberculosis results in the clinical presentation of mesenteric lymphadenitis. MLNs are important for immune responses to intestinal pathogens and microbiota in addition to their clinical relevance to Y. pseudotuberculosis infections. A characteristic of Y. pseudotuberculosis infection in MLNs is the formation of pyogranulomas. Pyogranulomas are composed of neutrophils, inflammatory monocytes, and lymphocytes surrounding extracellular microcolonies of Y. pseudotuberculosis. Key elements of the complex pathogen-host interaction in MLNs have been identified using mouse infection models. Y. pseudotuberculosis requires the virulence plasmid pYV to induce the formation of pyogranulomas in MLNs. The YadA adhesin and the Ysc-Yop type III secretion system (T3SS) are encoded on pYV. YadA mediates bacterial binding to host receptors, which engages the T3SS to preferentially translocate seven Yop effectors into phagocytes. The effectors promote pathogenesis by blocking innate immune defenses such as superoxide production, degranulation, and inflammasome activation, resulting in survival and growth of Y. pseudotuberculosis. On the other hand, certain effectors can trigger immune defenses in phagocytes. For example, YopJ triggers activation of caspase-8 and an apoptotic cell death response in monocytes within pyogranulomas that limits dissemination of Y. pseudotuberculosis from MLNs to the bloodstream. YopE can be processed as an antigen by phagocytes in MLNs, resulting in T and B cell responses to Y. pseudotuberculosis. Immune responses to Y. pseudotuberculosis in MLNs can also be detrimental to the host in the form of chronic lymphadenopathy. This review focuses on interactions between Y. pseudotuberculosis and phagocytes mediated by pYV that concurrently promote pathogenesis and host defense in MLNs. We propose that MLN pyogranulomas are immunological arenas in which opposing pYV-driven forces determine the outcome of infection in favor of the pathogen or host.

Copper is an essential micronutrient that also exerts toxic effects at high concentrations. This review summarizes the current state of knowledge on copper handling and homeostasis systems in Escherichia coli and Salmonella enterica. We describe the mechanisms by which transcriptional regulators, efflux pumps, detoxification enzymes, metallochaperones, and ancillary copper response systems orchestrate cellular response to copper stress. E. coli and S. enterica are important pathogens of humans and animals. We discuss the critical role of copper during killing of these pathogens by macrophages and in nutritional immunity at the bacterial-pathogen-host interface. In closing, we identify opportunities to advance our understanding of the biological roles of copper in these model enteric bacterial pathogens.

Iron is an essential element for Escherichia, Salmonella, and Shigella species. The acquisition of sufficient amounts of iron is difficult in many environments, including the intestinal tract, where these bacteria usually reside. Members of these genera have multiple iron transport systems to transport both ferrous and ferric iron. These include transporters for free ferrous iron, ferric iron associated with chelators, and heme. The numbers and types of transport systems in any species reflect the diversity of niches that it can inhabit. Many of the iron transport genes are found on mobile genetic elements or pathogenicity islands, and there is evidence of the spread of the genes among different species and pathotypes. This is notable among the pathogenic members of the genera in which iron transport systems acquired by horizontal gene transfer allow the bacteria to overcome host innate defenses that act to restrict the availability of iron to the pathogen. The need for iron is balanced by the need to avoid iron overload since excess iron is toxic to the cell. Genes for iron transport and metabolism are tightly regulated and respond to environmental cues, including iron availability, oxygen, and temperature. Master regulators, the iron sensor Fur and the Fur-regulated small RNA (sRNA) RyhB, coordinate the expression of iron transport and cellular metabolism genes in response to the availability of iron.

Proteins are major contributors to the composition and the functions in the cell. They often assemble into larger structures, macromolecular machines, to carry out intricate essential functions. Although huge progress in understanding how macromolecular machines function has been made by reconstituting them in vitro, the role of the intracellular environment is still emerging. The development of fluorescence microscopy techniques in the last 2 decades has allowed us to obtain an increased understanding of proteins and macromolecular machines in cells. Here, we describe how proteins move by diffusion, how they search for their targets, and how they are affected by the intracellular environment. We also describe how proteins assemble into macromolecular machines and provide examples of how frequent subunit turnover is used for them to function and to respond to changes in the intracellular conditions. This review emphasizes the constant movement of molecules in cells, the stochastic nature of reactions, and the dynamic nature of macromolecular machines.

Vitamin B₆ is an ensemble of six interconvertible vitamers: pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL), and their 5'-phosphate derivatives, PNP, PMP, and PLP. Pyridoxal 5'-phosphate is a coenzyme in a variety of enzyme reactions concerning transformations of amino and amino acid compounds. This review summarizes all known and putative PLP-binding proteins found in the Escherichia coli MG1655 proteome. PLP can have toxic effects since it contains a very reactive aldehyde group at its 4' position that easily forms aldimines with primary and secondary amines and reacts with thiols. Most PLP is bound either to the enzymes that use it as a cofactor or to PLP carrier proteins, protected from the cellular environment but at the same time readily transferable to PLP-dependent apoenzymes. E. coli and its relatives synthesize PLP through the seven-step deoxyxylulose-5-phosphate (DXP)-dependent pathway. Other bacteria synthesize PLP in a single step, through a so-called DXP-independent pathway. Although the DXP-dependent pathway was the first to be revealed, the discovery of the widespread DXP-independent pathway determined a decline of interest in E. coli vitamin B₆ metabolism. In E. coli, as in most organisms, PLP can also be obtained from PL, PN, and PM, imported from the environment or recycled from protein turnover, via a salvage pathway. Our review deals with all aspects of vitamin B₆ metabolism in E. coli, from transcriptional to posttranslational regulation. A critical interpretation of results is presented, in particular, concerning the most obscure aspects of PLP homeostasis and delivery to PLP-dependent enzymes.

As the spread of antibiotic resistance threatens our ability to treat infections, avoiding the return of a preantibiotic era requires the discovery of new drugs. While therapeutic use of antibiotics followed by the inevitable selection of resistance is a modern phenomenon, these molecules and the genetic determinants of resistance were in use by environmental microbes long before humans discovered them. In this review, we discuss evidence that antibiotics and resistance were present in the environment before anthropogenic use, describing techniques including direct sampling of ancient DNA and phylogenetic analyses that are used to reconstruct the past. We also pay special attention to the ecological and evolutionary forces that have shaped the natural history of antibiotic biosynthesis, including a discussion of competitive versus signaling roles for antibiotics, proto-resistance, and substrate promiscuity of biosynthetic and resistance enzymes. Finally, by applying an evolutionary lens, we describe concepts governing the origins and evolution of biosynthetic gene clusters and cluster-associated resistance determinants. These insights into microbes' use of antibiotics in nature, a game they have been playing for millennia, can provide inspiration for discovery technologies and management strategies to combat the growing resistance crisis.

Bacterial plasmids have been linked to virulence in Escherichia coli and Salmonella since their initial discovery. Though the plasmid repertoire of these bacterial species is extremely diverse, virulence-associated attributes tend to be limited to a small subset of plasmid types. This is particularly true for extraintestinal pathogenic E. coli, or ExPEC, where a handful of plasmids have been recognized to confer virulence- and fitness-associated traits. The purpose of this review is to highlight the biological and genomic attributes of ExPEC virulence-associated plasmids, with an emphasis on high-risk dominant ExPEC clones. Two specific plasmid types are highlighted to illustrate the independently evolved commonalities of these clones relative to plasmid content. Furthermore, the dissemination of these plasmids within and between bacterial species is examined. These examples demonstrate the evolution of high-risk clones toward common goals, and they show that rare transfer events can shape the ecological landscape of dominant clones within a pathotype.