Recombineering in Non-Model Bacteria.

The technology of recombineering, in vivo genetic engineering, was initially developed in Escherichia coli and uses bacteriophage-encoded homologous recombination proteins to efficiently recombine DNA at short homologies (35 to 50 nt). Because the technology is homology driven, genomic DNA can be modified precisely and independently of restriction site location. Recombineering uses linear DNA substrates that are introduced into the cell by electroporation; these can be PCR products, synthetic double-strand DNA (dsDNA), or single-strand DNA (ssDNA). Here we describe the applications, challenges, and factors affecting ssDNA and dsDNA recombineering in a variety of non-model bacteria, both Gram-negative and -positive, and recent breakthroughs in the field. We list different microbes in which the widely used phage λ Red and Rac RecET recombination systems have been used for in vivo genetic engineering. New homologous ssDNA and dsDNA recombineering systems isolated from non-model bacteria are also described. The Basic Protocol outlines a method for ssDNA recombineering in the non-model species of Shewanella. The Alternate Protocol describes the use of CRISPR/Cas as a counter-selection system in conjunction with recombineering to enhance recovery of recombinants. We provide additional background information, pertinent considerations for experimental design, and parameters critical for success. The design of ssDNA oligonucleotides (oligos) and various internet-based tools for oligo selection from genome sequences are also described, as is the use of oligo-mediated recombination. This simple form of genome editing uses only ssDNA oligo(s) and does not require an exogenous recombination system. The information presented here should help researchers identify a recombineering system suitable for their microbe(s) of interest. If no system has been characterized for a specific microbe, researchers can find guidance in developing a recombineering system from scratch. We provide a flowchart of decision-making paths for strategically applying annealase-dependent or oligo-mediated recombination in non-model and undomesticated bacteria. © 2022 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: ssDNA recombineering in Shewanella species Alternate Protocol: ssDNA recombineering coupled to CRISPR/Cas9 in Shewanella species.

Engineering Wired Life: Synthetic Biology for Electroactive Bacteria.

Electroactive bacteria produce or consume electrical current by moving electrons to and from extracellular acceptors and donors. This specialized process, known as extracellular electron transfer, relies on pathways composed of redox active proteins and biomolecules and has enabled technologies ranging from harvesting energy on the sea floor, to chemical sensing, to carbon capture. Harnessing and controlling extracellular electron transfer pathways using bioengineering and synthetic biology promises to heighten the limits of established technologies and open doors to new possibilities. In this review, we provide an overview of recent advancements in genetic tools for manipulating native electroactive bacteria to control extracellular electron transfer. After reviewing electron transfer pathways in natively electroactive organisms, we examine lessons learned from the introduction of extracellular electron transfer pathways into Escherichia coli. We conclude by presenting challenges to future efforts and give examples of opportunities to bioengineer microbes for electrochemical applications.

Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-Mediated Counter-Selection.

Dissimilatory metal-reducing bacteria, particularly those from the genus Shewanella, are of importance for bioremediation of metal contaminated sites and sustainable energy production. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulation. Here we report the development of a highly efficient system based on single-stranded DNA oligonucleotide recombineering coupled with CRISPR/Cas9-mediated counter-selection. Our system uses two plasmids: a sgRNA targeting vector and an editing vector, the latter harboring both Cas9 and the phage recombinase W3 Beta. Following the experimental analysis of Cas9 activity, we demonstrate the ability of this system to efficiently and precisely engineer different Shewanella strains with an average efficiency of >90% among total transformed cells, compared to ≃5% by recombineering alone, and regardless of the gene modified. We also show that different genetic changes can be introduced: mismatches, deletions, and small insertions. Surprisingly, we found that use of CRISPR/Cas9 alone allows selection of recombinase-independent S. oneidensis mutations, albeit at lower efficiency and frequency. With synthesized single-stranded DNA as substrates for homologous recombination and Cas9 as a counter-selectable marker, this new system provides a rapid, scalable, versatile, and scarless tool that will accelerate progress in Shewanella genomic engineering.

A new recombineering system for precise genome-editing in Shewanella oneidensis strain MR-1 using single-stranded oligonucleotides.

Shewanella oneidensis MR-1 is an invaluable host for the discovery and engineering of pathways important for bioremediation of toxic and radioactive metals and understanding extracellular electron transfer. However, genetic manipulation is challenging due to the lack of genetic tools. Previously, the only reliable method used for introducing DNA into Shewanella spp. at high efficiency was bacterial conjugation, enabling transposon mutagenesis and targeted knockouts using suicide vectors for gene disruptions. Here, we describe development of a robust and simple electroporation method in S. oneidensis that allows an efficiency of ~4.0 x 106 transformants/µg DNA. High transformation efficiency is maintained when cells are frozen for long term storage. In addition, we report a new prophage-mediated genome engineering (recombineering) system using a λ Red Beta homolog from Shewanella sp. W3-18-1. By targeting two different chromosomal alleles, we demonstrate its application for precise genome editing using single strand DNA oligonucleotides and show that an efficiency of ~5% recombinants among total cells can be obtained. This is the first effective and simple strategy for recombination with markerless mutations in S. oneidensis. Continued development of this recombinant technology will advance high-throughput and genome modification efforts to engineer and investigate S. oneidensis and other environmental bacteria.

[Preliminary multicentric study on the nutritional status in hospitalization at home (HaD) with the same informational application of nutritional assessment]. / Estudio multicéntrico preliminar sobre el estado nutricional en hospitalización a domicilio (HaD) con una misma aplicación informática de valoración nutricional.

INTRODUCTION: the prevalence of hospital malnutrition is high and involves an increase in health care costs. Home hospitalization (HH) allows better clinically stable patient control after an acute illness by a highly specialized health care team. OBJECTIVE: to know the nutritional condition of home hospitalization patients using a computer application that allows the implementation of early nutritional measures at home and improves post-hospital control of these patients. MATERIAL AND METHODS: prospective multicenter study of the nutritional condition of patients in four different home hospitalization centers during a period of two consecutive months in 2016. Variables were collected: home hospitalization, age, gender, reason for admission, associated morbidity, origin, diagnosis, social assessment, previous nutritional support, height, weight, weight loss, time of weight loss, total proteins, albumin, lymphocytes, cholesterol, body mass index (BMI), nutritional condition, type and degree of malnutrition. Nutritional condition was assessed using the application HEN-Persan and results were statistically analyzed using the SPSS 21.0 software. RESULTS: no significant differences were found between the four centers. In home hospitalization patients, 36% presented a normal nutritional screening and 87% presented some degree of malnutrition, while combined malnutrition prevailed (63%). Depending on the nutritional degree, 36% of patients had mild malnutrition, 27% presented moderate malnutrition and 35% had severe malnutrition. CONCLUSIONS: a computer application allows for an immediate, secure and reliable nutritional assessment in home hospitalization that helps introduce early nutritional measures and improve post-hospital control of patients.

INTRODUCCIÓN: la malnutrición hospitalaria tiene una elevada prevalencia y comporta un incremento del coste sanitario. La hospitalización a domicilio (HaD) permite el control en el domicilio de un episodio hospitalario agudo estable clínicamente por un equipo sanitario especializado.Objetivo: conocer el estado nutricional de los pacientes que ingresen en HaD con una misma aplicación informática (app) para poder instaurar medidas nutricionales precoces en el domicilio y mejorar la evolución posthospitalaria de los pacientes. MATERIAL Y METODOLOGÍA: estudio multicéntrico prospectivo y descriptivo del estado nutricional en cuatro unidades de HaD, durante un periodo de dos meses consecutivos durante el año 2016. Se recogieron las variables: unidad de HaD, edad, sexo, motivo de ingreso, patología asociada, procedencia, diagnóstico, valoración social, soporte nutricional previo, talla, peso, pérdida de peso, tiempo de la pérdida de peso, proteínas totales, albúmina, linfocitos, colesterol, índice de masa corporal (IMC), estado nutricional según el IMC, riesgo nutricional, tipo y grado de desnutrición. Se realizó la valoración nutricional con la app HEN-Persan y se analizaron los resultados estadísticamente con el programa informático SPSS 21.0. RESULTADOS: no existen diferencias significativas entre las cuatro unidades. Globalmente, el 36% de pacientes ingresados en HaD presentaban un estado nutricional normal. El 87% presentaba algún tipo de malnutrición, predominando la desnutrición de tipo mixta (63%). Según el grado,fue leve (36%), moderada (27%) y grave (35%). CONCLUSIONES: la utilización de una app permite tener una valoración nutricional inmediata, de manera fácil, segura y fiable en HaD, para poder introducir medidas nutricionales precoces y mejorar la evolución posthospitalaria de los pacientes.