A Model for Mechanical Stress Limited Bacterial Growth and Resporulation in Confinement.

In this study, we propose a self-limiting growth model forBacillus subtilisspores confined within porous polyacrylamide (PA) hydrogels. We observed thatB. subtilisspores germinate into vegetative cells within the hydrogel matrix, forming spherical colonies. These colonies expand until the mechanical stress they exert on their environment surpasses the yield stress of the hydrogel, leading to formation of a nonpermeable layer that halts nutrient diffusion and forces the bacteria to resporulate. These novel observations suggest a model to explain why bacterial growth in confined environments and material interfaces may be limited, providing insight for natural phenomena and biotechnological applications involving bacterial encapsulation.

Electron bifurcation and fluoride efflux systems implicated in defluorination of perfluorinated unsaturated carboxylic acids by Acetobacterium spp.

Enzymatic cleavage of C─F bonds in per- and polyfluoroalkyl substances (PFAS) is largely unknown but avidly sought to promote systems biology for PFAS bioremediation. Here, we report the reductive defluorination of α, ß-unsaturated per- and polyfluorocarboxylic acids by Acetobacterium spp. The microbial defluorination products were structurally confirmed and showed regiospecificity and stereospecificity, consistent with their formation by enzymatic reactions. A comparison of defluorination activities among several Acetobacterium species indicated that a functional fluoride exporter was required for the detoxification of the released fluoride. Results from both in vivo inhibition tests and in silico enzyme modeling suggested the involvement of enzymes of the flavin-based electron-bifurcating caffeate reduction pathway [caffeoyl-CoA reductase (CarABCDE)] in the reductive defluorination. This is a report on specific microorganisms carrying out enzymatic reductive defluorination of PFAS, which could be linked to electron-bifurcating reductases that are environmentally widespread.

The link between ancient microbial fluoride resistance mechanisms and bioengineering organofluorine degradation or synthesis.

Fluorinated organic chemicals, such as per- and polyfluorinated alkyl substances (PFAS) and fluorinated pesticides, are both broadly useful and unusually long-lived. To combat problems related to the accumulation of these compounds, microbial PFAS and organofluorine degradation and biosynthesis of less-fluorinated replacement chemicals are under intense study. Both efforts are undermined by the substantial toxicity of fluoride, an anion that powerfully inhibits metabolism. Microorganisms have contended with environmental mineral fluoride over evolutionary time, evolving a suite of detoxification mechanisms. In this perspective, we synthesize emerging ideas on microbial defluorination/fluorination and fluoride resistance mechanisms and identify best approaches for bioengineering new approaches for degrading and making organofluorine compounds.

Microwell fluoride assay screening for enzymatic defluorination.

There is intense interest in removing fluorinated compounds from the environment, environments are most efficiently remediated by microbial enzymes, and defluorinating enzymes are readily monitored by fluoride determination. Fluorine is the most electronegative element. Consequently, all mechanisms of enzymatic C-F bond cleavage produce fluoride anion, F-. Therefore, methods for the determination of fluoride are critical for C-F enzymology and apply to any fluorinated organic compounds, including PFAS, or per- and polyfluorinated alkyl substances. The biodegradation of most PFAS chemicals is rare or unknown. Accordingly, identifying new enzymes, or re-engineering the known defluorinases, will require rapid and sensitive methods for measuring fluoride in aqueous media. Most studies currently use ion chromatography or fluoride specific electrodes which are relatively sensitive but low throughput. The methods here describe refashioning a drinking water test to efficiently determine fluoride in enzyme and cell culture reaction mixtures. The method is based on lanthanum alizarin complexone binding of fluoride. Reworking the method to a microtiter well plate format allows detection of as little as 4 nmol of fluoride in 200 µL of assay buffer. The method is amenable to color imaging, spectrophotometric plate reading and automated liquid handling to expedite assays with thousands of enzymes and/or substrates for discovering and improving enzymatic defluorination.

Evolutionary obstacles and not C-F bond strength make PFAS persistent.

The fate of organic matter in the environment, including anthropogenic chemicals, is largely predicated on the enzymatic capabilities of microorganisms. Microbes readily degrade, and thus recycle, most of the ~100,000 commercial chemicals used in modern society. Per- and polyfluorinated compounds (PFAS) are different. Many research papers posit that the general resistance of PFAS to microbial degradation is based in chemistry and that argument relates to the strength of the C-F bond. Here, I advance the opinion that the low biodegradability of PFAS is best formulated as a biological optimization problem, hence evolution. The framing of the problem is important. If it is framed around C-F bond strength, the major effort should focus on finding and engineering new C-F cleaving enzymes. The alternative, and preferred approach suggested here, is to focus on the directed evolution of biological systems containing known C-F cleaving systems. There are now reports of bacteria degrading and/or growing on multiply fluorinated arenes, alkenoic and alkanoic acids. The impediment to more efficient and widespread biodegradation in these systems is biological, not chemical. The rationale for this argument is made in the five sections below that follow the Introduction.

Insights into the action of the pharmaceutical metformin: Targeted inhibition of the gut microbial enzyme agmatinase.

Metformin is the first-line treatment for type 2 diabetes, yet its mechanism of action is not fully understood. Recent studies suggest metformin's interactions with gut microbiota are responsible for exerting therapeutic effects. In this study, we report that metformin targets the gut microbial enzyme agmatinase, as a competitive inhibitor, which may impair gut agmatine catabolism. The metformin inhibition constant (Ki) of E. coli agmatinase is 1 mM and relevant in the gut where the drug concentration is 1-10 mM. Metformin analogs phenformin, buformin, and galegine are even more potent inhibitors of E. coli agmatinase (Ki = 0.6, 0.1, and 0.007 mM, respectively) suggesting a shared mechanism. Agmatine is a known effector of human host metabolism and has been reported to augment metformin's therapeutic effects for type 2 diabetes. This gut-derived inhibition mechanism gives new insights on metformin's action in the gut and may lead to significant discoveries in improving metformin therapy.

Dinickel enzyme evolved to metabolize the pharmaceutical metformin and its implications for wastewater and human microbiomes.

Metformin is the first-line treatment for type II diabetes patients and a pervasive pollutant with more than 180 million kg ingested globally and entering wastewater. The drug's direct mode of action is currently unknown but is linked to effects on gut microbiomes and may involve specific gut microbial reactions to the drug. In wastewater treatment plants, metformin is known to be transformed by microbes to guanylurea, although genes encoding this metabolism had not been elucidated. In the present study, we revealed the function of two genes responsible for metformin decomposition (mfmA and mfmB) found in isolated bacteria from activated sludge. MfmA and MfmB form an active heterocomplex (MfmAB) and are members of the ureohydrolase protein superfamily with binuclear metal-dependent activity. MfmAB is nickel-dependent and catalyzes the hydrolysis of metformin to dimethylamine and guanylurea with a catalytic efficiency (kcat/KM) of 9.6 × 103 M-1s-1 and KM for metformin of 0.82 mM. MfmAB shows preferential activity for metformin, being able to discriminate other close substrates by several orders of magnitude. Crystal structures of MfmAB show coordination of binuclear nickel bound in the active site of the MfmA subunit but not MfmB subunits, indicating that MfmA is the active site for the MfmAB complex. Mutagenesis of residues conserved in the MfmA active site revealed those critical to metformin hydrolase activity and its small substrate binding pocket allowed for modeling of bound metformin. This study characterizes the products of the mfmAB genes identified in wastewater treatment plants on three continents, suggesting that metformin hydrolase is widespread globally in wastewater.

Recombinant Pseudomonas growing on non-natural fluorinated substrates shows stress but overall tolerance to cytoplasmically released fluoride anion.

IMPORTANCE: Society uses thousands of organofluorine compounds, sometimes denoted per- and polyfluoroalkyl substances (PFAS), in hundreds of products, but recent studies have shown some to manifest human and environmental health effects. As a class, they are recalcitrant to biodegradation, partly due to the paucity of fluorinated natural products to which microbes have been exposed. Another limit to PFAS biodegradation is the intracellular toxicity of fluoride anion generated from C-F bond cleavage. The present study identified a broader substrate specificity in an enzyme originally studied for its activity on the natural product fluoroacetate. A recombinant Pseudomonas expressing this enzyme was used here as a model system to better understand the limits and effects of a high level of intracellular fluoride generation. A fluoride stress response has evolved in bacteria and has been described in Pseudomonas spp. The present study is highly relevant to organofluorine compound degradation or engineered biosynthesis in which fluoride anion is a substrate.

A microbial evolutionary approach for a sustainable future.

With the continued population increase, more sustainable use of water, land, air and chemicals is imperative. Microorganisms will need to be called upon to aid in many sustainability efforts. Prokaryotes are the fastest-evolving cellular life, and most manipulatable via synthetic biology. Moreover, their natural diversity in processing organic and inorganic chemicals, and their survivability in extreme niches, make them prime agents to enlist for solving many of society's pressing problems.