More Like this

1. Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform

   https://doi.org/10.1038/s41467-022-32727-w  

   Zhang, Linyue ; King, Edward ; Black, William B. ; Heckmann, Christian M. ; Wolder, Allison ; Cui, Youtian ; Nicklen, Francis ; Siegel, Justin B. ; Luo, Ray ; Paul, Caroline E. ; et al ( August 2022 , Nature Communications)

   Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)⁺) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN⁺). We achieve this by engineering the life-essential glutathione reductase inEscherichia colito exclusively rely on the reduced NMN⁺(NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN⁺with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA⁺), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)⁺.
2. Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes

   https://doi.org/10.1186/s40168-022-01454-1  

   Kleiner, Manuel ; Kouris, Angela ; Violette, Marlene ; D’Angelo, Grace ; Liu, Yihua ; Korenek, Abigail ; Tolić, Nikola ; Sachsenberg, Timo ; McCalder, Janine ; Lipton, Mary S. ; et al ( February 2023 , Microbiome)

   Stable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates, as well as the activity of species. The application of these approaches ranges from studying microbial communities important for global biogeochemical cycling to host-microbiota interactions in the intestinal tract. Current SIP approaches, such as DNA-SIP or nanoSIMS allow to analyze incorporation of stable isotopes with high coverage of taxa in a community and at the single cell level, respectively, however they are limited in terms of sensitivity, resolution or throughput.

   Here, we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by 50–99% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. The approach allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics liquid chromatography-tandem mass spectrometry (LC–MS/MS) measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software (https://sourceforge.net/projects/calis-p/). We demonstrate sensitivity, precision and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, we benchmarkmore »our approach against two existing Protein-SIP approaches and show that in the low labeling range used our approach is the most sensitive and accurate. Finally, we measure translational activity using¹⁸O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were severalBacteroidesspecies known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics.

   We demonstrate that our Protein-SIP approach allows for the ultra-sensitive (0.01 to 10% label) detection of stable isotopes of elements found in proteins, using standard metaproteomics data.

   « less
3. The rate and fate of N2 and C fixation by marine diatom-diazotroph symbioses

   https://doi.org/10.1038/s41396-021-01086-7  

   Foster, Rachel A. ; Tienken, Daniela ; Littmann, Sten ; Whitehouse, Martin J. ; Kuypers, Marcel M. M. ; White, Angelicque E. ( August 2021 , The ISME Journal)

   N₂fixation constitutes an important new nitrogen source in the open sea. One group of filamentous N₂fixing cyanobacteria (Richelia intracellularis, hereafterRichelia)form symbiosis with a few genera of diatoms. High rates of N₂fixation and carbon (C) fixation have been measured in the presence of diatom-Richeliasymbioses. However, it is unknown how partners coordinate C fixation and how the symbiont sustains high rates of N₂fixation. Here, both the N₂and C fixation in wild diatom-Richeliapopulations are reported. Inhibitor experiments designed to inhibit host photosynthesis, resulted in lower estimated growth and depressed C and N₂fixation, suggesting that despite the symbionts ability to fix their own C, they must still rely on their respective hosts for C. Single cell analysis indicated that up to 22% of assimilated C in the symbiont is derived from the host, whereas 78–91% of the host N is supplied from their symbionts. A size-dependent relationship is identified where larger cells have higher N₂and C fixation, and only N₂fixation was light dependent. Using the single cell measures, the N-rich phycosphere surrounding these symbioses was estimated and contributes directly and rapidly to the surface ocean rather than the mesopelagic, even at high estimated sinking velocities (<10 m d⁻¹). Several eco-physiological parameters necessary for incorporatingmore »symbiotic N₂fixing populations into larger basin scale biogeochemical models (i.e., N and C cycles) are provided.

   « less
4. Early detection and classification of live bacteria using time-lapse coherent imaging and deep learning

   https://doi.org/10.1038/s41377-020-00358-9  

   Wang, Hongda ; Ceylan Koydemir, Hatice ; Qiu, Yunzhe ; Bai, Bijie ; Zhang, Yibo ; Jin, Yiyin ; Tok, Sabiha ; Yilmaz, Enis Cagatay ; Gumustekin, Esin ; Rivenson, Yair ; et al ( July 2020 , Light: Science & Applications)

   Early identification of pathogenic bacteria in food, water, and bodily fluids is very important and yet challenging, owing to sample complexities and large sample volumes that need to be rapidly screened. Existing screening methods based on plate counting or molecular analysis present various tradeoffs with regard to the detection time, accuracy/sensitivity, cost, and sample preparation complexity. Here, we present a computational live bacteria detection system that periodically captures coherent microscopy images of bacterial growth inside a 60-mm-diameter agar plate and analyses these time-lapsed holograms using deep neural networks for the rapid detection of bacterial growth and the classification of the corresponding species. The performance of our system was demonstrated by the rapid detection ofEscherichia coliand total coliform bacteria (i.e.,Klebsiella aerogenesandKlebsiella pneumoniaesubsp. pneumoniae) in water samples, shortening the detection time by >12 h compared to the Environmental Protection Agency (EPA)-approved methods. Using the preincubation of samples in growth media, our system achieved a limit of detection (LOD) of ~1 colony forming unit (CFU)/L in ≤9 h of total test time. This platform is highly cost-effective (~$0.6/test) and has high-throughput with a scanning speed of 24 cm²/min over the entire plate surface, making it highly suitable for integration with the existing methods currently usedmore »for bacteria detection on agar plates. Powered by deep learning, this automated and cost-effective live bacteria detection platform can be transformative for a wide range of applications in microbiology by significantly reducing the detection time and automating the identification of colonies without labelling or the need for an expert.

   « less
5. Implications of high rates of sexual recruitment in driving rapid reef recovery in Mo’orea, French Polynesia

   https://doi.org/10.1038/s41598-018-34686-z  

   Edmunds, Peter J. ( November 2018 , Scientific Reports)

   Coral abundance continues to decline on tropical reefs around the world, and this trend suggests that coral reefs may not persist beyond the current century. In contrast, this study describes the near-complete mortality of corals on the outer reef (10 m and 17 m depth) of the north shore of Mo’orea, French Polynesia, from 2005 to 2010, followed by unprecedented recovery from 2011 to 2017. Intense corallivory and a cyclone drove coral cover from 33–48% to <3% by 2010, but over the following seven years, recovery occurred through rapid population growth (up to 12% cover y⁻¹) to 25–74% cover by 2017. The thirteen-year, U-shape trajectory of coral cover over time created by the loss and replacement of millions of corals through sexual reproduction underscores the potential for beneficial genetic responses to environmental conditions for at least one genus,Pocillopora. The high ecological resilience of this coral community appears to have been enhanced by variation among genera in the susceptibility to declining cover, and the capacity for population growth (i.e., response diversity). These results suggest that the outer coral communities of Mo’orea may be poised for genetic changes that could affect their capacity to persistence.