Ruminococcus bromii enables the growth of proximal Bacteroides thetaiotaomicron by releasing glucose during starch degradation.

Complex carbohydrates shape the gut microbiota, and the collective fermentation of resistant starch by gut microbes positively affects human health through enhanced butyrate production. The keystone species Ruminococcus bromii (Rb) is a specialist in degrading resistant starch; its degradation products are used by other bacteria including Bacteroides thetaiotaomicron (Bt). We analysed the metabolic and spatial relationships between Rb and Bt during potato starch degradation and found that Bt utilizes glucose that is released from Rb upon degradation of resistant potato starch and soluble potato amylopectin. Additionally, we found that Rb produces a halo of glucose around it when grown on solid media containing potato amylopectin and that Bt cells deficient for growth on potato amylopectin (∆sus Bt) can grow within the halo. Furthermore, when these ∆sus Bt cells grow within this glucose halo, they have an elongated cell morphology. This long-cell phenotype depends on the glucose concentration in the solid media: longer Bt cells are formed at higher glucose concentrations. Together, our results indicate that starch degradation by Rb cross-feeds other bacteria in the surrounding region by releasing glucose. Our results also elucidate the adaptive morphology of Bt cells under different nutrient and physiological conditions.

New Orange Ligand-Dependent Fluorescent Reporter for Anaerobic Imaging.

Bilin-binding fluorescent proteins like UnaG-bilirubin are noncovalent ligand-dependent reporters for oxygen-free microscopy but are restricted to blue and far-red fluorescence. Here we describe a high-throughput screening approach to provide a new UnaG-ligand pair that can be excited in the 532 nm green excitation microscopy channel. We identified a novel orange UnaG-ligand pair that maximally emits at 581 nm. Whereas the benzothiazole-based ligand itself is nominally fluorescent, the compound binds UnaG with high affinity (Kd = 3 nM) to induce a 2.5-fold fluorescence intensity enhancement and a 10 nm red shift. We demonstrated this pair in the anaerobic fluorescence microscopy of the prevalent gut bacterium Bacteroides thetaiotaomicron and in Escherichia coli. This UnaG-ligand pair can also be coupled to IFP2.0-biliverdin to differentiate cells in mixed-species two-color imaging. Our results demonstrate the versatility of the UnaG ligand-binding pocket and extend the ability to image cells at longer wavelengths in anoxic environments.

Imaging living obligate anaerobic bacteria with bilin-binding fluorescent proteins.

Fluorescent tools such as green fluorescent protein (GFP) have been used extensively as reporters in biochemistry and microbiology, but GFP and other conventional fluorescent proteins are restricted to aerobic environments. This limitation precludes fluorescence studies of anaerobic ecologies including polymicrobial communities in the human gut microbiome and in soil microbiomes, which profoundly affect health, disease, and the environment. To address this limitation, we describe the first implementation of two bilin-binding fluorescent proteins (BBFPs), UnaG and IFP2.0, as oxygen-independent fluorescent labels for live-cell imaging in anaerobic bacteria. Expression of UnaG or IFP2.0 in the prevalent gut bacterium Bacteroides thetaiotaomicron (B. theta) results in detectable fluorescence upon the addition of the bilirubin or biliverdin ligand, even in anaerobic conditions. Furthermore, these BBFPs can be used in two-color imaging to differentiate cells expressing either UnaG or IFP2.0; UnaG and IFP2.0 can also be used to distinguish B. theta from other common gut bacterial species in mixed-culture live-cell imaging. BBFPs are promising fluorescent tools for live-cell imaging investigations of otherwise inaccessible anaerobic polymicrobial communities.

Extending fluorescence microscopy into anaerobic environments.

Fluorescence microscopy is a powerful tool for investigating living cells. While widely used fluorescent proteins, such as green fluorescent protein (GFP), have had huge impact in biological imaging because they provide genetically encoded, highly specific labeling, these probes require oxygen to generate fluorescence. This crucial oxidative step has limited the use of GFP-like proteins in anaerobic bacterial systems and restricted live-cell studies of obligate anaerobes and their biology. This review discusses alternative approaches to labeling proteins in anaerobic bacteria that are compatible with live-cell fluorescence microscopy in strict oxygen-free environments. The advantages, disadvantages, and likelihood of successful implementation for each approach are considered to provide context and guide further advances in anaerobic fluorescence labeling.

Design and Discovery of New Combinations of Mutant DNA Polymerases and Modified DNA Substrates.

Chemical modifications can enhance the properties of DNA by imparting nuclease resistance and generating more-diverse physical structures. However, native DNA polymerases generally cannot synthesize significant lengths of DNA with modified nucleotide triphosphates. Previous efforts have identified a mutant of DNA polymerase I from Thermus aquaticus DNA (SFM19) as capable of synthesizing a range of short, 2'-modified DNAs; however, it is limited in the length of the products it can synthesize. Here, we rationally designed and characterized ten mutants of SFM19. From this, we identified enzymes with substantially improved activity for the synthesis of 2'F-, 2'OH-, 2'OMe-, and 3'OMe-modified DNA as well as for reverse transcription of 2'OMe DNA. We also evaluated mutant DNA polymerases previously only tested for synthesis for 2'OMe DNA and showed that they are capable of an expanded range of modified DNA synthesis. This work significantly expands the known combinations of modified DNA and Taq DNA polymerase mutants.

Taq DNA Polymerase Mutants and 2'-Modified Sugar Recognition.

Chemical modifications to DNA, such as 2' modifications, are expected to increase the biotechnological utility of DNA; however, these modified forms of DNA are limited by their inability to be effectively synthesized by DNA polymerase enzymes. Previous efforts have identified mutant Thermus aquaticus DNA polymerase I (Taq) enzymes capable of recognizing 2'-modified DNA nucleotides. While these mutant enzymes recognize these modified nucleotides, they are not capable of synthesizing full length modified DNA; thus, further engineering is required for these enzymes. Here, we describe comparative biochemical studies that identify useful, but previously uncharacterized, properties of these enzymes; one enzyme, SFM19, is able to recognize a range of 2'-modified nucleotides much wider than that previously examined, including fluoro, azido, and amino modifications. To understand the molecular origins of these differences, we also identify specific amino acids and combinations of amino acids that contribute most to the previously evolved unnatural activity. Our data suggest that a negatively charged amino acid at 614 and mutation of the steric gate residue, E615, to glycine make up the optimal combination for modified oligonucleotide synthesis. These studies yield an improved understanding of the mutational origins of 2'-modified substrate recognition as well as identify SFM19 as the best candidate for further engineering, whether via rational design or directed evolution.