Roles of Proteins Containing Immunoglobulin-Like Domains in the Conjugation of Bacterial Plasmids

Transmission of a plasmid from one bacterial cell to another, in several instances, underlies the dissemination of antimicrobial resistance (AMR) genes. The process requires well-characterized enzymatic machinery that facilitates cell-to-cell contact and the transfer of the plasmid.

A Parallelized Nanofluidic Device for High-Throughput Optical DNA Mapping of Bacterial Plasmids

Optical DNA mapping (ODM) has developed into an important technique for DNA analysis, where single DNA molecules are sequence-specifically labeled and stretched, for example, in nanofluidic channels. We have developed an ODM assay to analyze bacterial plasmids—circular extrachromosomal DNA that often carry genes that make bacteria resistant to antibiotics. As for most techniques, the next important step is to increase throughput and automation. In this work, we designed and fabricated a nanofluidic device that, together with a simple automation routine, allows parallel analysis of up to 10 samples at the same time. Using plasmids encoding extended-spectrum beta-lactamases (ESBL), isolated from Escherichia coli and Klebsiella pneumoniae, we demonstrate the multiplexing capabilities of the device when it comes to both many samples in parallel and different resistance genes. As a final example, we combined the device with a novel protocol for rapid cultivation and extraction of plasmids from fecal samples collected from patients. This combined protocol will make it possible to analyze many patient samples in one device already on the day the sample is collected, which is an important step forward for the ODM analysis of plasmids in clinical diagnostics.

Roles of proteins containing immunoglobulin-like domains in the conjugation of bacterial plasmids.

Horizontal transfer of bacterial plasmids generates genetic variability and contributes to the dissemination of the genes that enable bacterial cells to develop antimicrobial resistance (AMR). Several aspects of the conjugative process have long been known, namely, those related to the proteins that participate in the establishment of cell-to-cell contact and to the enzymatic processes associated with the processing of plasmid DNA and its transfer to the recipient cell. In this work, we describe the role of newly identified proteins that influence the conjugation of several plasmids. Genes encoding high-molecular-weight proteins that contain one or several immunoglobulin-like domains (Big) are located in the transfer regions of several plasmids that usually harbor AMR determinants. These Big proteins are exported to the external medium and target two extracellular organelles: the flagella and the conjugative pili. The plasmid-encoded Big proteins facilitate conjugation by reducing cell motility (by binding to flagella) and facilitating cell-to-cell contact (by binding to the conjugative pilus). They use the same export machinery as that used by the conjugative pilus components. In the examples characterized in this paper, these proteins influence conjugation at environmental temperatures (i.e., 25C). This suggests that they may play relevant roles in the dissemination of plasmids in natural environments. As they are located in outer surface organelles, they could be targeted to control the dissemination of different bacterial plasmids carrying AMR determinants.

An Overview of the Importance of Bacterial Elements for Plant Genetic Engineering

Plant genetic engineering is one of the most important aspects of biotechnology applied to plant systems. The stable introduction of exogenous genetic material in plant cells is a determining step for obtaining transgenic plants. In this context, bacteria are crucial for the development of transgenic plants. Gene cloning often involves the use of bacterial plasmids and DNAmodifying enzymes synthesized in genetically modified bacteria. In addition, among the several methods of introducing genes into plants, the method that uses Agrobacterium tumefaciens continues to be used to obtain genetically modified plants for the agricultural, pharmaceutical and materials industry sectors. This minireview aims to present the basic aspects of bacterial elements related gene manipulation for obtaining transgenic plants.

Toxin-Antidote Elements Across the Tree of Life

In life's constant battle for survival, it takes one to kill but two to conquer. Toxin-antitoxin or toxin-antidote (TA) elements are genetic dyads that cheat the laws of inheritance to guarantee their transmission to the next generation. This seemingly simple genetic arrangement—a toxin linked to its antidote—is capable of quickly spreading and persisting in natural populations. TA elements were first discovered in bacterial plasmids in the 1980s and have recently been characterized in fungi, plants, and animals, where they underlie genetic incompatibilities and sterility in crosses between wild isolates. In this review, we provide a unified view of TA elements in both prokaryotic and eukaryotic organisms and highlight their similarities and differences at the evolutionary, genetic, and molecular levels. Finally, we propose several scenarios that could explain the paradox of the evolutionary origin of TA elements and argue that these elements may be key evolutionary players and that the full scope of their roles is only beginning to be uncovered.

SynBio and the Boundaries between Functional and Pathogenic RepA-WH1 Bacterial Amyloids

ABSTRACT Amyloids are protein polymers that were initially linked to human diseases. Across the whole Tree of Life, many disease-unrelated proteins are now emerging for which amyloids represent distinct functional states. Most bacterial amyloids described are extracellular, contributing to biofilm formation. However, only a few have been found in the bacterial cytosol. This paper reviews from the perspective of synthetic biology (SynBio) our understanding of the subtle line that separates functional from pathogenic and transmissible amyloids (prions). In particular, it is focused on RepA-WH1, a functional albeit unconventional natural amyloidogenic protein domain that participates in controlling DNA replication of bacterial plasmids. SynBio approaches, including protein engineering and the design of allosteric effectors such as diverse ligands and an optogenetic module, have enabled the generation in RepA-WH1 of an intracellular cytotoxic prion-like agent in bacteria. The synthetic RepA-WH1 prion has the potential to develop into novel antimicrobials.