Extracting and characterizing protein-free megabase-pair DNA for in vitro experiments

Chromosome structure and function is studied using various cell-based methods as well as with a range of in vitro single-molecule techniques on short DNA substrates. Here, we present a method to obtain megabase-pair-length deproteinated DNA for in vitro studies. We isolated chromosomes from bacterial cells and enzymatically digested the native proteins. Mass spectrometry indicated that 97%–100% of DNA-binding proteins are removed from the sample. Fluorescence microscopy analysis showed an increase in the radius of gyration of the DNA polymers, while the DNA length remained megabase-pair sized. In proof-of-concept experiments using these deproteinated long DNA molecules, we observed DNA compaction upon adding the DNA-binding protein Fis or PEG crowding agents and showed that it is possible to track the motion of a fluorescently labeled DNA locus. These results indicate the practical feasibility of a “genome-in-a-box” approach to study chromosome organization from the bottom up.BN/Cees Dekker LabDynamics of Micro and Nano SystemsBT/Environmental Biotechnolog

A Novel D-Galacturonate Fermentation Pathway in Lactobacillus suebicus Links Initial Reactions of the Galacturonate-Isomerase Route With the Phosphoketolase Pathway

D-galacturonate, a key constituent of pectin, is a ubiquitous monomer in plant biomass. Anaerobic, fermentative conversion of D-galacturonate is therefore relevant in natural environments as well as in microbial processes for microbial conversion of pectin-containing agricultural residues. In currently known microorganisms that anaerobically ferment D-galacturonate, its catabolism occurs via the galacturonate-isomerase pathway. Redox-cofactor balancing in this pathway strongly constrains the possible range of products generated from anaerobic D-galacturonate fermentation, resulting in acetate as the predominant organic fermentation product. To explore metabolic diversity of microbial D-galacturonate fermentation, anaerobic enrichment cultures were performed at pH 4. Anaerobic batch and chemostat cultures of a dominant Lactobacillus suebicus strain isolated from these enrichment cultures produced near-equimolar amounts of lactate and acetate from D-galacturonate. A combination of whole-genome sequence analysis, quantitative proteomics, enzyme activity assays in cell extracts, and in vitro product identification demonstrated that D-galacturonate metabolism in L. suebicus occurs via a novel pathway. In this pathway, mannonate generated by the initial reactions of the canonical isomerase pathway is converted to 6-phosphogluconate by two novel biochemical reactions, catalyzed by a mannonate kinase and a 6-phosphomannonate 2-epimerase. Further catabolism of 6-phosphogluconate then proceeds via known reactions of the phosphoketolase pathway. In contrast to the classical isomerase pathway for D-galacturonate catabolism, the novel pathway enables redox-cofactor-neutral conversion of D-galacturonate to ribulose-5-phosphate. While further research is required to identify the structural genes encoding the key enzymes for the novel pathway, its redox-cofactor coupling is highly interesting for metabolic engineering of microbial cell factories for conversion of pectin-containing feedstocks into added-value fermentation products such as ethanol or lactate. This study illustrates the potential of microbial enrichment cultivation to identify novel pathways for the conversion of environmentally and industrially relevant compounds.BT/Industrial MicrobiologyOLD BT/Cell Systems EngineeringBT/Environmental BiotechnologyBT/Biotechnolog

Cellular assays identify barriers impeding iron-sulfur enzyme activity in a non-native prokaryotic host

Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile. However, Fe-S proteins and enzymes are often inactive outside their native host species. We sought to systematically investigate the compatibility of Fe-S networks with non-native Fe-S enzymes. By using collections of Fe-S enzyme orthologs representative of the entire range of prokaryotic diversity, we uncovered a striking correlation between phylogenetic distance and probability of functional expression. Moreover, coexpression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be supported by the foreign host. We also find that Fe-S enzymes that require specific electron carrier proteins are rarely functionally expressed unless their taxon-specific reducing partners are identified and co-expressed. We demonstrate how these principles can be applied to improve the activity of a radical S-adenosyl methionine(rSAM) enzyme from a Streptomyces antibiotic biosynthesis pathway in Escherichia coli. Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineering functional Fe-S enzyme-dependent pathways.BN/Greg Bokinsky LabBT/Environmental Biotechnolog

Using a 29-mRNA Host Response Classifier To Detect Bacterial Coinfections and Predict Outcomes in COVID-19 Patients Presenting to the Emergency Department

Clinicians in the emergency department (ED) face challenges in concurrently assessing patients with suspected COVID-19 infection, detecting bacterial coinfection, and determining illness severity since current practices require separate workflows. Here, we explore the accuracy of the IMX-BVN-3/IMX-SEV-3 29 mRNA host response classifiers in simultaneously detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and bacterial coinfections and predicting clinical severity of COVID-19. A total of 161 patients with PCR-confirmed COVID-19 (52.2% female; median age, 50.0 years; 51% hospitalized; 5.6% deaths) were enrolled at the Stanford Hospital ED. RNA was extracted (2.5 mL whole blood in PAXgene blood RNA), and 29 host mRNAs in response to the infection were quantified using Nanostring nCounter. The IMX-BVN-3 classifier identified SARS-CoV-2 infection in 151 patients with a sensitivity of 93.8%. Six of 10 patients undetected by the classifier had positive COVID tests more than 9 days prior to enrollment, and the remaining patients oscillated between positive and negative results in subsequent tests. The classifier also predicted that 6 (3.7%) patients had a bacterial coinfection. Clinical adjudication confirmed that 5/6 (83.3%) of the patients had bacterial infections, i.e., Clostridioides difficile colitis (n = 1), urinary tract infection (n = 1), and clinically diagnosed bacterial infections (n = 3), for a specificity of 99.4%. Two of 101 (2.8%) patients in the IMX-SEV-3 "Low" severity classification and 7/60 (11.7%) in the "Moderate" severity classification died within 30 days of enrollment. IMX-BVN-3/IMX-SEV-3 classifiers accurately identified patients with COVID-19 and bacterial coinfections and predicted patients' risk of death. A point-of-care version of these classifiers, under development, could improve ED patient management, including more accurate treatment decisions and optimized resource utilization. IMPORTANCE We assay the utility of the single-test IMX-BVN-3/IMX-SEV-3 classifiers that require just 2.5 mL of patient blood in concurrently detecting viral and bacterial infections as well as predicting the severity and 30-day outcome from the infection. A point-of-care device, in development, will circumvent the need for blood culturing and drastically reduce the time needed to detect an infection. This will negate the need for empirical use of broad-spectrum antibiotics and allow for antibiotic use stewardship. Additionally, accurate classification of the severity of infection and the prediction of 30-day severe outcomes will allow for appropriate allocation of hospital resources