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1. Cas9-assisted biological containment of a genetically engineered human commensal bacterium and genetic elements

   https://doi.org/10.1038/s41467-024-45893-w  

   Hayashi, Naoki ; Lai, Yong ; Fuerte-Stone, Jay ; Mimee, Mark ; Lu, Timothy K. ( March 2024 , Nature Communications)

   Sophisticated gene circuits built by synthetic biology can enable bacteria to sense their environment and respond predictably. Engineered biosensing bacteria outfitted with such circuits can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. To provide robust biocontainment for engineered bacteria, we devised a Cas9-assisted auxotrophic biocontainment system combining thymidine auxotrophy, an Engineered Riboregulator (ER) for controlled gene expression, and a CRISPR Device (CD). The CD prevents the engineered bacteria from acquiringthyAvia horizontal gene transfer, which would disrupt the biocontainment system, and inhibits the spread of genetic elements by killing bacteria harboring the gene cassette. This system tunably controlled gene expression in the human gut commensal bacteriumBacteroides thetaiotaomicron, prevented escape from thymidine auxotrophy, and blocked transgene dissemination. These capabilities were validated in vitro and in vivo. This biocontainment system exemplifies a powerful strategy for bringing genetically engineered microorganisms safely into biomedicine.

    

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2. Design and implementation of an affordable laboratory‐scale bioreactor for the production of microbial natural products

   https://doi.org/10.1002/eng2.12059  

   Theodore, Christine M. ; Loveridge, Steven T. ; Crews, Mitchell S. ; Lorig‐Roach, Nicholas ; Crews, Phillip ( November 2019 , Engineering Reports)

   Large‐scale bacteria culturing can present many challenges for small academic laboratories. To address these challenges a cost effective, laboratory scale bioreactor was designed and implemented. The constructed bioreactor addresses common problems that small or teaching‐focused laboratories face when attempting scale up cultures. The design utilizes materials commonly found in standard chemistry laboratories that are easily assembled with minor modifications. The system was validated through the replication of natural product production in shake flasks and the bioreactor. Additionally, measurements were done to ensure the designed bioreactor had comparablek_Lavalues to common shake flask conditions. We anticipate that this design will be of use to other small academic natural product groups as well as teaching laboratories as it offers an economical way to undertake large scale culturing of microorganisms.

    

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3. Multiplexed microfluidic screening of bacterial chemotaxis

   https://doi.org/10.7554/eLife.85348  

   Stehnach, Michael R ; Henshaw, Richard J ; Floge, Sheri A ; Guasto, Jeffrey S ( July 2023 , eLife)

   Microorganism sensing of and responding to ambient chemical gradients regulates a myriad of microbial processes that are fundamental to ecosystem function and human health and disease. The development of efficient, high-throughput screening tools for microbial chemotaxis is essential to disentangling the roles of diverse chemical compounds and concentrations that control cell nutrient uptake, chemorepulsion from toxins, and microbial pathogenesis. Here, we present a novel microfluidic multiplexed chemotaxis device (MCD) which uses serial dilution to simultaneously perform six parallel bacterial chemotaxis assays that span five orders of magnitude in chemostimulant concentration on a single chip. We first validated the dilution and gradient generation performance of the MCD, and then compared the measured chemotactic response of an established bacterial chemotaxis system (Vibrio alginolyticus) to a standard microfluidic assay. Next, the MCD’s versatility was assessed by quantifying the chemotactic responses of different bacteria (Psuedoalteromonas haloplanktis, Escherichia coli) to different chemoattractants and chemorepellents. The MCD vastly accelerates the chemotactic screening process, which is critical to deciphering the complex sea of chemical stimuli underlying microbial responses.

    

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4. Gallionellaceae in rice root plaque: metabolic roles in iron oxidation, nutrient cycling, and plant interactions

   https://doi.org/10.1128/aem.00570-23  

   Chan, Clara S. ; Dykes, Gretchen E. ; Hoover, Rene L. ; Limmer, Matt A. ; Seyfferth, Angelia L. ( December 2023 , Applied and Environmental Microbiology)

   Glass, Jennifer B. (Ed.)

   On the roots of wetland plants such as rice, Fe(II) oxidation forms Fe(III) oxyhydroxide-rich plaques that modulate plant nutrient and metal uptake. The microbial roles in catalyzing this oxidation have been debated and it is unclear if these iron-oxidizers mediate other important biogeochemical and plant interactions. To investigate this, we studied the microbial communities, metagenomes, and geochemistry of iron plaque on field-grown rice, plus the surrounding rhizosphere and bulk soil. Plaque iron content (per mass root) increased over the growing season, showing continuous deposition. Analysis of 16S rRNA genes showed abundant Fe(II)-oxidizing and Fe(III)-reducing bacteria (FeOB and FeRB) in plaque, rhizosphere, and bulk soil. FeOB were enriched in relative abundance in plaque, suggesting FeOB affinity for the root surface. Gallionellaceae FeOBSideroxydanswere enriched during vegetative and early reproductive rice growth stages, while aGallionellawas enriched during reproduction through grain maturity, suggesting distinct FeOB niches over the rice life cycle. FeRBAnaeromyxobacterandGeobacterincreased in plaque later, during reproduction and grain ripening, corresponding to increased plaque iron. Metagenome-assembled genomes revealed that Gallionellaceae may grow mixotrophically using both Fe(II) and organics. TheSideroxydansare facultative, able to use non-Fe substrates, which may allow colonization of rice roots early in the season. FeOB genomes suggest adaptations for interacting with plants, including colonization, plant immunity defense, utilization of plant organics, and nitrogen fixation. Taken together, our results strongly suggest that rhizoplane and rhizosphere FeOB can specifically associate with rice roots, catalyzing iron plaque formation, with the potential to contribute to plant growth.

   In waterlogged soils, iron plaque forms a reactive barrier between the root and soil, collecting phosphate and metals such as arsenic and cadmium. It is well established that iron-reducing bacteria solubilize iron, releasing these associated elements. In contrast, microbial roles in plaque formation have not been clear. Here, we show that there is a substantial population of iron oxidizers in plaque, and furthermore, that these organisms (SideroxydansandGallionella) are distinguished by genes for plant colonization and nutrient fixation. Our results suggest that iron-oxidizing and iron-reducing bacteria form and remodel iron plaque, making it a dynamic system that represents both a temporary sink for elements (P, As, Cd, C, etc.) as well as a source. In contrast to abiotic iron oxidation, microbial iron oxidation results in coupled Fe-C-N cycling, as well as microbe-microbe and microbe-plant ecological interactions that need to be considered in soil biogeochemistry, ecosystem dynamics, and crop management.

    

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5. Unusual Boronium Salt Shows Antifungal Activity Comparable to a Commercial Quaternary Ammonium Disinfectant

   https://doi.org/10.1002/slct.202104344  

   Ravine, Terrence J. ; Soltani, Mohammad ; Davis, Jr., James H. ; Salter, Edward A. ; Wierzbicki, Andrzej ( March 2022 , ChemistrySelect)

   Previously, a boronium salt possessing a terminal benzyl group was reported to have greater antibacterial activity than a commercial quaternary ammonium disinfectant solution againstEscherichia coli,Pseudomonas aeruginosa, andStaphylococcus aureus. Results of the current study indicate that the same boronium salt without a benzyl group, exhibited equal or better antifungal activity against actively growingCandida albicansyeast andAspergillus fumigatusmold when compared to the same quat disinfectant. This same compound also displayed antifungal activity against dormantA. fumigatusspores comparable to the quat disinfectant. In contrast, the boronium ion with a benzyl group was 4–16X less effective than either the non‐benzylated form or quat disinfectant for all 3 fungal test conditions. The observation that the boronium salt without a benzyl group exhibited substantial antifungal activity in the current study but did not display any antibacterial activity in the previous study is of particular interest. This finding represents a flip‐flop outcome from the previous bacterial testing. It suggests that the presence of a terminal benzyl group greatly influences the boronium ion's ability to interact with fungal membranes.

    

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