Twin-arginine translocase component TatB performs folding quality control via a chaperone-like activity.

The twin-arginine translocation (Tat) pathway involves an inbuilt quality control (QC) system that synchronizes the proofreading of substrate protein folding with lipid bilayer transport. However, the molecular details of this QC mechanism remain poorly understood. Here, we hypothesized that the conformational state of Tat substrates is directly sensed by the TatB component of the bacterial Tat translocase. In support of this hypothesis, several TatB variants were observed to form functional translocases in vivo that had compromised QC activity as evidenced by the uncharacteristic export of several misfolded protein substrates. These variants each possessed cytoplasmic membrane-extrinsic domains that were either truncated or mutated in the vicinity of a conserved, highly flexible α-helical domain. In vitro folding experiments revealed that the TatB membrane-extrinsic domain behaved like a general molecular chaperone, transiently binding to highly structured, partially unfolded intermediates of a model protein, citrate synthase, in a manner that prevented its irreversible aggregation and stabilized the active species. Collectively, these results suggest that the Tat translocase may use chaperone-like client recognition to monitor the conformational status of its substrates.

Two-Tiered Selection and Screening Strategy to Increase Functional Enzyme Production in E. coli.

Development of recombinant enzymes as industrial biocatalysts or metabolic pathway elements requires soluble expression of active protein. Here we present a two-step strategy, combining a directed evolution selection with an enzyme activity screen, to increase the soluble production of enzymes in the cytoplasm of E. coli. The directed evolution component relies on the innate quality control of the twin-arginine translocation pathway coupled with antibiotic selection to isolate point mutations that promote intracellular solubility. A secondary screen is applied to ensure the solubility enhancement has not compromised enzyme activity. This strategy has been successfully applied to increase the soluble production of a fungal endocellulase by 30-fold in E. coli without change in enzyme specific activity through two rounds of directed evolution.

Engineering a Supersecreting Strain of Escherichia coli by Directed Coevolution of the Multiprotein Tat Translocation Machinery.

Escherichia coli remains one of the preferred hosts for biotechnological protein production due to its robust growth in culture and ease of genetic manipulation. It is often desirable to export recombinant proteins into the periplasmic space for reasons related to proper disulfide bond formation, prevention of aggregation and proteolytic degradation, and ease of purification. One such system for expressing heterologous secreted proteins is the twin-arginine translocation (Tat) pathway, which has the unique advantage of delivering correctly folded proteins into the periplasm. However, transit times for proteins through the Tat translocase, comprised of the TatABC proteins, are much longer than for passage through the SecYEG pore, the translocase associated with the more widely utilized Sec pathway. To date, a high protein flux through the Tat pathway has yet to be demonstrated. To address this shortcoming, we employed a directed coevolution strategy to isolate mutant Tat translocases for their ability to deliver higher quantities of heterologous proteins into the periplasm. Three supersecreting translocases were selected that each exported a panel of recombinant proteins at levels that were significantly greater than those observed for wild-type TatABC or SecYEG translocases. Interestingly, all three of the evolved Tat translocases exhibited quality control suppression, suggesting that increased translocation flux was gained by relaxation of substrate proofreading. Overall, our discovery of more efficient translocase variants paves the way for the use of the Tat system as a powerful complement to the Sec pathway for secreted production of both commodity and high value-added proteins.

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells.

Glycans and glycosylated biomolecules are directly involved in almost every biological process as well as the etiology of most major diseases. Hence, glycoscience knowledge is essential to efforts aimed at addressing fundamental challenges in understanding and improving human health, protecting the environment and enhancing energy security, and developing renewable and sustainable resources that can serve as the source of next-generation materials. While much progress has been made, there remains an urgent need for new tools that can overexpress structurally uniform glycans and glycoconjugates in the quantities needed for characterization and that can be used to mechanistically dissect the enzymatic reactions and multi-enzyme assembly lines that promote their construction. To address this technology gap, cell-free synthetic glycobiology has emerged as a simplified and highly modular framework to investigate, prototype, and engineer pathways for glycan biosynthesis and biomolecule glycosylation outside the confines of living cells. From nucleotide sugars to complex glycoproteins, we summarize here recent efforts that harness the power of cell-free approaches to design, build, test, and utilize glyco-enzyme reaction networks that produce desired glycomolecules in a predictable and controllable manner. We also highlight novel cell-free methods for shedding light on poorly understood aspects of diverse glycosylation processes and engineering these processes toward desired outcomes. Taken together, cell-free synthetic glycobiology represents a promising set of tools and techniques for accelerating basic glycoscience research (e.g., deciphering the "glycan code") and its application (e.g., biomanufacturing high-value glycomolecules on demand).

Developmental stage influences chromosome segregation patterns and arrangement in the extremely polyploid, giant bacterium Epulopiscium sp. type B.

Few studies have described chromosomal dynamics in bacterial cells with more than two complete chromosome copies or described changes with respect to development in polyploid cells. We examined the arrangement of chromosomal loci in the very large, highly polyploid, uncultivated intestinal symbiont Epulopiscium sp. type B using fluorescent in situ hybridization. We found that in new offspring, chromosome replication origins (oriCs) are arranged in a three-dimensional array throughout the cytoplasm. As development progresses, most oriCs become peripherally located. Siblings within a mother cell have similar numbers of oriCs. When chromosome orientation was assessed in situ by labeling two chromosomal regions, no specific pattern was detected. The Epulopiscium genome codes for many of the conserved positional guide proteins used for chromosome segregation in bacteria. Based on this study, we present a model that conserved chromosomal maintenance proteins, combined with entropic demixing, provide the forces necessary for distributing oriCs. Without the positional regulation afforded by radial confinement, chromosomes are more randomly oriented in Epulopiscium than in most small rod-shaped cells. Furthermore, we suggest that the random orientation of individual chromosomes in large polyploid cells would not hamper reproductive success as it would in smaller cells with more limited genomic resources.

Characterization of MocR, a GntR-like transcriptional regulator, in Bradyrhizobium japonicum: its impact on motility, biofilm formation, and soybean nodulation.

Bradyrhizobium japonicum is a Gram-negative soil bacterium that can fix nitrogen into ammonia by developing a symbiotic relationship with the soybean plant. MocR proteins make up a subfamily of GntR superfamily, one of the most widely distributed and prolific groups of the helix-turn-helix transcription factors. In this study, we constructed a mutant strain for mocR (blr6977) to investigate its role in cellular processes and symbiosis in B. japonicum. Although growth rate and morphology of the mutant were indistinguishable from those of the wild type, the mutant showed significant differences in motility and attachment (i.e., biofilm formation) from the wild type. The mutant displayed a decrease in biofilm formation, but was more motile than the wild type. The inactivation of mocR did not affect the number of nodules on soybean roots, but caused delayed nodulation. Delayed nodulation intrigued us to study competitiveness of the mutant infecting soybeans. The mutant was less competitive than the wild type, indicating that delayed nodulation might be due to competitiveness. Gene expressions of other MocR subfamily members were also compared between the wild type and mutant strains. None of the mocR-like genes examined in this study were differentially expressed between both strains.

Repurposing a bacterial quality control mechanism to enhance enzyme production in living cells.

Heterologous expression of many proteins in bacteria, yeasts, and plants is often limited by low titers of functional protein. To address this problem, we have created a two-tiered directed evolution strategy in Escherichia coli that enables optimization of protein production while maintaining high biological activity. The first tier involves a genetic selection for intracellular protein stability that is based on the folding quality control mechanism inherent to the twin-arginine translocation pathway, while the second is a semi-high-throughput screen for protein function. To demonstrate the utility of this strategy, we isolated variants of the endoglucanase Cel5A, from the plant-pathogenic fungus Fusarium graminearum, whose production was increased by as much as 30-fold over the parental enzyme. This gain in production was attributed to just two amino acid substitutions, and it was isolated after two iterations through the two-tiered approach. There was no significant tradeoff in activity on soluble or insoluble cellulose substrates. Importantly, by combining the folding filter afforded by the twin-arginine translocation quality control mechanism with a function-based screen, we show enrichment for variants with increased protein abundance in a manner that does not compromise catalytic activity, providing a highly soluble parent for engineering of improved or new function.

Beneficial regulation of matrixmetalloproteinases and their inhibitors, fibrillar collagens and transforming growth factor-beta by Polypodium leucotomos, directly or in dermal fibroblasts, ultraviolet radiated fibroblasts, and melanoma cells.

The extracellular matrix (ECM) that gives tissue its structural integrity is remodeled in skin aging/photoaging and cancer via the increased expression/activities of matrixmetalloproteinases (MMP), inhibition of the tissue inhibitors of matrix metalloproteinases (TIMP), or inhibition of collagen synthesis. Transforming growth factor-beta (TGF-beta), a predominant regulator of the ECM, is inhibited in aging/photoaging and stimulated in carcinogenesis. P. leucotomos (fern) extract has potential to counteract these alterations via its antioxidant, anti-inflammatory and photoprotective properties. The goal of this research was to determine the efficacy of P. leucotomos to (a) directly inhibit MMP-1, 2, 3, and 9 activities, (b) inhibit MMP-2, and stimulate TIMPs, fibrillar collagens and TGF-beta in non-irradiated or ultraviolet (UV) radiated fibroblasts, and (c) inhibit MMPs and TGF-beta, and stimulate TIMPs in melanoma cells. To this purpose, we examined the direct effect of P. leucotomos (0-1%) on MMPs' activities, and its effects on the expression (protein and/or transcription levels) of (1) MMPs and TIMPs in dermal fibroblasts, and melanoma cells, (2) TGF-beta in non-irradiated, UVA (2.5 J/cm2) or UVB (2.5 mJ/cm2) irradiated fibroblasts, and melanoma cells, and (3) types I, III, and V collagen in non-irradiated or UV irradiated fibroblasts. P. leucotomos directly inhibited the activities of MMPs as well as the expression of MMPs in fibroblasts, and melanoma cells while stimulating the expression of TIMPs in these cells. P. leucotomos stimulated types I, III, and V collagen in non-irradiated fibroblasts, and types I and V collagen in UV radiated fibroblasts. P. leucotomos had predominant stimulatory effects on TGF-beta expression in non-irradiated or UV radiated fibroblasts, and inhibited TGF-beta expression in melanoma cells. The effects of P. leucotomos were largely similar to that of ascorbic acid. P. leucotomos demonstrated dual protective effects on the ECM via its inhibition of the ECM proteolytic enzymes and the stimulation of the structural ECM collagens. The effects of P. leucotomos on fibroblasts and melanoma cells may be partly via its cell-specific regulation of TGF-beta expression and partly via its antioxidant property. The intake or topical application of P. leucotomos may be beneficial to skin health, in aging and cancer prevention or treatment.