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Microbiology and synthetic biology depend on reverse genetic approaches to manipulate bacterial genomes; however, existing methods require molecular biology to generate genomic homology, suffer from low efficiency, and are not easily scaled to high throughput. To overcome these limitations, we developed a system for creating kilobase-scale genomic modifications that uses DNA oligonucleotides to direct the integration of a non-replicating plasmid. This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting (ORBIT) was pioneered in Mycobacteria, and here we adapt and expand it for Escherichia coli. Our redesigned plasmid toolkit for oligonucleotide recombineering achieved significantly higher efficiency than λ Red double-stranded DNA recombineering and enabled precise, stable knockouts (≤134 kb) and integrations (≤11 kb) of various sizes. Additionally, we constructed multi-mutants in a single transformation, using orthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides to knock out nearly all known transcription factor and small RNA genes, yielding accurate, genome-wide, single mutant libraries. By counting genomic barcodes, we also show ORBIT libraries can scale to thousands of unique members (>30k). This work demonstrates that ORBIT for E. coli is a flexible reverse genetic system that facilitates rapid construction of complex strains and readily scales to create sophisticated mutant libraries., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered the biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tsypin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A strain of Geobacter sulfurreducens, an organism capable of respiring solid extracellular substrates, lacking four of five outer membrane cytochrome complexes ( extABCD + strain) grows faster and produces greater current density than the wild type grown under identical conditions. To understand cellular and biofilm modifications in the extABCD + strain responsible for this increased performance, biofilms grown using electrodes as terminal electron acceptors were sectioned and imaged using electron microscopy to determine changes in thickness and cell density, while parallel biofilms incubated in the presence of nitrogen and carbon isotopes were analyzed using NanoSIMS (nanoscale secondary ion mass spectrometry) to quantify and localize anabolic activity. Long-distance electron transfer parameters were measured for wild-type and extABCD + biofilms spanning 5-μm gaps. Our results reveal that extABCD + biofilms achieved higher current densities through the additive effects of denser cell packing close to the electrode (based on electron microscopy), combined with higher metabolic rates per cell compared to the wild type (based on increased rates of 15 N incorporation). We also observed an increased rate of electron transfer through extABCD + versus wild-type biofilms, suggesting that denser biofilms resulting from the deletion of unnecessary multiheme cytochromes streamline electron transfer to electrodes. The combination of imaging, physiological, and electrochemical data confirms that engineered electrogenic bacteria are capable of producing more current per cell and, in combination with higher biofilm density and electron diffusion rates, can produce a higher final current density than the wild type. IMPORTANCE Current-producing biofilms in microbial electrochemical systems could potentially sustain technologies ranging from wastewater treatment to bioproduction of electricity if the maximum current produced could be increased and current production start-up times after inoculation could be reduced. Enhancing the current output of microbial electrochemical systems has been mostly approached by engineering physical components of reactors and electrodes. Here, we show that biofilms formed by a Geobacter sulfurreducens strain producing ∼1.4× higher current than the wild type results from a combination of denser cell packing and higher anabolic activity, enabled by an increased rate of electron diffusion through the biofilms. Our results confirm that it is possible to engineer electrode-specific G. sulfurreducens strains with both faster growth on electrodes and streamlined electron transfer pathways for enhanced current production.

Redox cycling of extracellular electron shuttles can enable the metabolic activity of subpopulations within multicellular bacterial biofilms that lack direct access to electron acceptors or donors. How these shuttles catalyze extracellular electron transfer (EET) within biofilms without being lost to the environment has been a long-standing question. Here, we show that phenazines mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by eDNA binding. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and can participate directly in redox reactions through DNA. In vivo, biofilm eDNA can also support rapid electron transfer between redox active intercalators. Together, these results establish that PYO:eDNA interactions support an efficient redox cycle with rapid EET that is faster than the rate of PYO loss from the biofilm., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We grew a soil enrichment culture to identify organisms that anaerobically oxidize phenazine-1-carboxylic acid. A strain of Citrobacter portucalensis was isolated from this enrichment and sequenced by both Illumina and PacBio technologies. It has a genome with a length of 5.3 Mb, a G+C content of 51.8%, and at least one plasmid., (Copyright © 2020 Tsypin et al.)

Extracellular electron transfer (EET) allows microbes to drive their metabolism through interactions with minerals or electrodes. In recent work, Light et al. (2018) discover a specialized EET pathway in Listeria monocytogenes with homologs in pathogens and gut commensals, suggesting that EET plays important roles in diverse environments., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Descriptions of the changeable, striking colors associated with secreted natural products date back well over a century. These molecules can serve as extracellular electron shuttles (EESs) that permit microbes to access substrates at a distance. In this review, we argue that the colorful world of EESs has been too long neglected. Rather than simply serving as a diagnostic attribute of a particular microbial strain, redox-active natural products likely play fundamental, underappreciated roles in the biology of their producers, particularly those that inhabit biofilms. Here, we describe the chemical diversity and potential distribution of EES producers and users, discuss the costs associated with their biosynthesis, and critically evaluate strategies for their economical usage. We hope this review will inspire efforts to identify and explore the importance of EES cycling by a wide range of microorganisms so that their contributions to shaping microbial communities can be better assessed and exploited.

In a study of 194 re-operations for retinal detachment in which one-third of the cases were managed without drainage of subretinal fluid, it was found that a number of failures occurred after non-drainage. Analysis of these failures shows that more than half could have been avoided if the correct choice of cases suitable for management without drainage of subretinal fluid had been made. It is felt that with careful attention to the criteria which are used to decide on the choice of surgical technique, the results obtained by the non-drainage method should be as good in the more complicated case as they have already been shown to be in the primary case. Because the drainage of subretinal fluid carries with it a risk of surgical complications and these complications may contribute to the development of massive preretinal retraction, management of the re-operation should be carried out without drainage of subretinal fluid whenever possible.

In 200 cases of retinal detachment successfully treated without drainage of subretinal fluid, complete reattachment of the retina was achieved in the first post-operative week in 76% of cases. Delay in subretinal fluid absorption in the remaining 24% of cases was directly related to the duration of the retinal detachment, but was not influenced by the patients age, refractive error or the characteristics of the detachment.

In 200 cases of retinal detachment successfully treated without drainage of subretinal fluid complete reattachment of the retina was achieved in the first postoperative week in 76 per cent of cases. Delay in subretinal fluid absorption in the remaining 24 per cent of cases was directly related to the duration of the retinal detachment but was not influenced by the patient's age, refractive error, or the characteristics of the detachment.

In a study of 194 reoperation in which one third of the cases were managed without drainage of SRF, it was found that a number of failures occurred after non-drainage. Analysis of these failures shows that more than half could have been avoided if the choice of operation had been correct. It is felt that with careful attention to the criteria which was used to decide on the choice of surgical technique, the results obtained by the non-drainage method should be as good in the more complicated case as they have already been shown to be in the primary case. Because the drainage of SRF carries with it a high risk of surgical complications and these complications frequently result in the development of MPR, management of the reoperation should be carried out without drainage of SRF whenever possible.

1. Adenocarcinoma in this series is commoner than squamous cell carcinoma. 2. Adenocarcinoma is a disease mainly affecting the ethmoid sinuses whereas squamous cell carcinoma is a disease mainly affecting the maxillary sinuses. 3. Patients with adenocarcinoma survive longer than those with squamous cell carcinoma. 4. Death from adenocarcinoma usually results from intra-cranial infiltration and therefore widespread resection with the assistance of a neuro-surgeon may be needed.

A case is presented of sudden deafness following cardio-pulmonary bypass surgery. Microembolism is believed to be the most likely cause as this is common following the use of pump oxygenator systems. There was no response to anticoagulants.