Transcriptome architecture of the three main lineages of agrobacteria.

Agrobacteria are a diverse, polyphyletic group of prokaryotes with multipartite genomes capable of transferring DNA into the genomes of host plants, making them an essential tool in plant biotechnology. Despite their utility in plant transformation, genome-wide transcriptional regulation is not well understood across the three main lineages of agrobacteria. Transcription start sites (TSSs) are a necessary component of gene expression and regulation. In this study, we used differential RNA-seq and a TSS identification algorithm optimized on manually annotated TSS, then validated with existing TSS to identify thousands of TSS with nucleotide resolution for representatives of each lineage. We extend upon the 356 TSSs previously reported in Agrobacterium fabrum C58 by identifying 1,916 TSSs. In addition, we completed genomes and phenotyping of Rhizobium rhizogenes C16/80 and Allorhizobium vitis T60/94, identifying 2,650 and 2,432 TSSs, respectively. Parameter optimization was crucial for an accurate, high-resolution view of genome and transcriptional dynamics, highlighting the importance of algorithm optimization in genome-wide TSS identification and genomics at large. The optimized algorithm reduced the number of TSSs identified internal and antisense to the coding sequence on average by 90.5% and 91.9%, respectively. Comparison of TSS conservation between orthologs of the three lineages revealed differences in cell cycle regulation of ctrA as well as divergence of transcriptional regulation of chemotaxis-related genes when grown in conditions that simulate the plant environment. These results provide a framework to elucidate the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. IMPORTANCE Transcription start sites (TSSs) are fundamental for understanding gene expression and regulation. Agrobacteria, a group of prokaryotes with the ability to transfer DNA into the genomes of host plants, are widely used in plant biotechnology. However, the genome-wide transcriptional regulation of agrobacteria is not well understood, especially in less-studied lineages. Differential RNA-seq and an optimized algorithm enabled identification of thousands of TSSs with nucleotide resolution for representatives of each lineage. The results of this study provide a framework for elucidating the mechanistic basis and evolution of pathology across the three main lineages of agrobacteria. The optimized algorithm also highlights the importance of parameter optimization in genome-wide TSS identification and genomics at large.

The pGinger Family of Expression Plasmids.

The pGinger suite of expression plasmids comprises 43 plasmids that will enable precise constitutive and inducible gene expression in a wide range of Gram-negative bacterial species. Constitutive vectors are composed of 16 synthetic constitutive promoters upstream of red fluorescent protein (RFP), with a broad-host-range BBR1 origin and a kanamycin resistance marker. The family also has seven inducible systems (Jungle Express, Psal/NahR, Pm/XylS, Prha/RhaS, LacO1/LacI, LacUV5/LacI, and Ptet/TetR) controlling RFP expression on BBR1/kanamycin plasmid backbones. For four of these inducible systems (Jungle Express, Psal/NahR, LacO1/LacI, and Ptet/TetR), we created variants that utilize the RK2 origin and spectinomycin or gentamicin selection. Relevant RFP expression and growth data have been collected in the model bacterium Escherichia coli as well as Pseudomonas putida. All pGinger vectors are available via the Joint BioEnergy Institute (JBEI) Public Registry. IMPORTANCE Metabolic engineering and synthetic biology are predicated on the precise control of gene expression. As synthetic biology expands beyond model organisms, more tools will be required that function robustly in a wide range of bacterial hosts. The pGinger family of plasmids constitutes 43 plasmids that will enable both constitutive and inducible gene expression in a wide range of nonmodel Proteobacteria.

Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production.

Despite advances in understanding the metabolism of Pseudomonas putida KT2440, a promising bacterial host for producing valuable chemicals from plant-derived feedstocks, a strain capable of producing free fatty acid-derived chemicals has not been developed. Guided by functional genomics, we engineered P. putida to produce medium- and long-chain free fatty acids (FFAs) to titers of up to 670 mg/L. Additionally, by taking advantage of the varying substrate preferences of paralogous native fatty acyl-CoA ligases, we employed a strategy to control FFA chain length that resulted in a P. putida strain specialized in producing medium-chain FFAs. Finally, we demonstrate the production of oleochemicals in these strains by synthesizing medium-chain fatty acid methyl esters, compounds useful as biodiesel blending agents, in various media including sorghum hydrolysate at titers greater than 300 mg/L. This work paves the road to produce high-value oleochemicals and biofuels from cheap feedstocks, such as plant biomass, using this host.

The Medial Gastrocnemius Recession, an Alternative Surgical Treatment for Isolated Gastrocnemius Contracture: A Cadaver Study With Discussion Emphasizing Variable Conjoint Tendon Anatomy.

BACKGROUND: Gastrocnemius recession is a popular procedure utilized to treat chronic conditions related to isolated gastrocnemius contracture (IGC). Recent anatomical research detailing variable gastrocsoleus tendon morphology has raised important questions regarding the safety of some traditional recession procedures. Alternative gastrocnemius recession strategies may produce comparable dorsiflexion improvement results while avoiding the surgical risk related to conjoint tendon anatomical variability. METHODS: Ten matched cadaver pairs were randomized to receive either a medial gastrocnemius recession (MGR) procedure or a gastrocnemius intramuscular recession "Baumann" procedure. Postoperative dorsiflexion improvement was measured and then compared between groups. Detailed postoperative surgical dissections were performed to assess structures at risk, conjoint tendon morphology, and anatomical symmetry. RESULTS: Medial gastrocnemius recession and Baumann procedures were equally effective at producing significant increases in passive ankle dorsiflexion. No sural nerve injuries were observed. Thirty-five percent of specimens showed direct muscular fusion of at least a portion of the distal gastrocnemius muscular tissue to the adjacent soleus. CONCLUSION: The MGR procedure produced comparable dorsiflexion improvement results to the Baumann procedure in our cadaver model. Surgeons must account for certain conjoint tendon anatomical variants when surgically treating IGC as traditional recession methods risk tendo-Achilles overlengthening. LEVELS OF EVIDENCE: Level V: Cadaver Study.

Tibiotalocalcaneal Arthrodesis with Structural Allograft for Management of Large Osseous Defects of the Hindfoot and Ankle: A Systematic Review and Meta-Analysis.

Large osseous defects of the hindfoot and ankle pose a surgical challenge. Tibiotalocalcaneal (TTC) arthrodesis utilizing a structural allograft may be required to fill the osseous void, preserve limb length and achieve fusion. Several authors have reported small case series on this topic, however outcomes have varied and no systematic review of this data has been published to date. The primary aim of this study is to report rates of osseous union, limb salvage and complications in patients undergoing TTC arthrodesis with a structural allograft. A total of 11 publications were identified that met the inclusion criteria. One hundred seventy-five patients were included with a weighted mean age of 60.5 (range 50-72) years and follow-up period of 29.7 (range 3-62) months. Femoral head allograft was the most commonly utilized structural graft and a retrograde intramedullary nail was the most common fixation construct. Results demonstrated an overall union rate of 67.4%, limb salvage rate of 92.5% and complication rate of 26.6%. Allograft-related complications were rare with an allograft fracture rate of 0.1% and allograft collapse rate of 1.2%. There was no significant difference in union rate when using a retrograde intramedullary nail versus a plate construct (p = .9148). TTC arthrodesis with use of a structural allograft is a viable treatment option for limb salvage when faced with complex hindfoot and ankle pathology involving large osseous defects. Despite high rates of radiographic nonunion, this approach can provide patients with a stable and functional limb while avoiding amputation.

Soft Tissue Reconstruction and Osteotomies for Pes Planovalgus Correction.

The correction of the flexible pes planovalgus foot and ankle is a complicated and somewhat controversial topic. After conservative methods fail, there is a wide range of possible soft tissue and bony procedures. The appropriate work up and understanding of the pathomechanics are vital to the correct choice of procedures to correct these deformities. Once the work up and procedure selection are done, the operation must also be technically performed well and with efficiency, as most often the condition is corrected with a variety of procedures. This article discusses some of the most common procedures necessary to fully correct the pes planovalgus foot and discusses the authors' technique and pearls.

Nitrogen Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing.

Pseudomonas putida KT2440 has long been studied for its diverse and robust metabolisms, yet many genes and proteins imparting these growth capacities remain uncharacterized. Using pooled mutant fitness assays, we identified genes and proteins involved in the assimilation of 52 different nitrogen containing compounds. To assay amino acid biosynthesis, 19 amino acid drop-out conditions were also tested. From these 71 conditions, significant fitness phenotypes were elicited in 672 different genes including 100 transcriptional regulators and 112 transport-related proteins. We divide these conditions into 6 classes, and propose assimilatory pathways for the compounds based on this wealth of genetic data. To complement these data, we characterize the substrate range of three promiscuous aminotransferases relevant to metabolic engineering efforts in vitro. Furthermore, we examine the specificity of five transcriptional regulators, explaining some fitness data results and exploring their potential to be developed into useful synthetic biology tools. In addition, we use manifold learning to create an interactive visualization tool for interpreting our BarSeq data, which will improve the accessibility and utility of this work to other researchers. IMPORTANCE Understanding the genetic basis of P. putida's diverse metabolism is imperative for us to reach its full potential as a host for metabolic engineering. Many target molecules of the bioeconomy and their precursors contain nitrogen. This study provides functional evidence linking hundreds of genes to their roles in the metabolism of nitrogenous compounds, and provides an interactive tool for visualizing these data. We further characterize several aminotransferases, lactamases, and regulators, which are of particular interest for metabolic engineering.

Draft Genome Sequence of Mycobacterium sp. Strain JC1 DSM 3803.

Mycobacterium sp. strain JC1 DSM 3803 is one of the few known bacteria predicted to possess the xylulose monophosphate (XuMP) pathway of C1 assimilation. The draft genome is 7,921,603 bp with a GC content of 66.88% and will allow more in-depth investigation of this bacterium's unique metabolism.

Accuracy of Weightbearing CT Scans for Patient-Specific Instrumentation in Total Ankle Arthroplasty.

BACKGROUND: Total ankle arthroplasty (TAA) is a popular and viable option for end-stage ankle arthritis. Posttraumatic arthritis is the most common etiology of ankle arthritis, which creates the additional challenge of osseus deformity. Accuracy and reproducibility in placing the implant on the mechanical axis has been shown to be paramount in all joint arthroplasty including total ankle replacement. Patient-specific preoperative navigation is a relatively new technology for TAA, and up until this past year has been based off of nonweightbearing (NWBCT) or simulated weightbearing computed tomography (WBCT). Our institution has created a protocol to use WBCT in the preoperative patient-specific navigation for TAA using the Prophecy system. The purpose of our study was to compare the accuracy and reproducibility of implant alignment and size using WBCT vs prior studies using NWBCT for the Prophecy reports. METHODS: All patients from July 2019 through October 2020 who underwent TAA were evaluated. Inclusion criteria consisted of primary TAA using patient-specific preoperative navigation who had postoperative radiographs in the 4-6-week time frame. Prophecy predictions and measurements were then compared to actual implant placement and size. RESULTS: Ten patients met our inclusion criteria of WBCT Prophecy preoperative planning using 2 different implant systems. Preoperative deformities in this cohort were small. The average postoperative coronal alignment was 0.84 degrees, range 0.19 to 2.4 degrees. Average postoperative sagittal plane deformity was 1.9 degrees, range 0.33 to 5.05 degrees. Tibial component size was properly predicted in all patients, talar component in 9 of 10. CONCLUSION: This initial report supports accuracy and reproducibility in preoperative patient-specific navigation when using WBCT for TAA with these implants. All TAAs were within the intended target of less than 5 degrees varus or valgus. LEVEL OF EVIDENCE: Level III, retrospective comparative analysis.

Investigation of Bar-seq as a method to study population dynamics of Saccharomyces cerevisiae deletion library during bioreactor cultivation.

BACKGROUND: Despite the latest advancements in metabolic engineering for genome editing and characterization of host performance, the successful development of robust cell factories used for industrial bioprocesses and accurate prediction of the behavior of microbial systems, especially when shifting from laboratory-scale to industrial conditions, remains challenging. To increase the probability of success of a scale-up process, data obtained from thoroughly performed studies mirroring cellular responses to typical large-scale stimuli may be used to derive crucial information to better understand potential implications of large-scale cultivation on strain performance. This study assesses the feasibility to employ a barcoded yeast deletion library to assess genome-wide strain fitness across a simulated industrial fermentation regime and aims to understand the genetic basis of changes in strain physiology during industrial fermentation, and the corresponding roles these genes play in strain performance. RESULTS: We find that mutant population diversity is maintained through multiple seed trains, enabling large scale fermentation selective pressures to act upon the community. We identify specific deletion mutants that were enriched in all processes tested in this study, independent of the cultivation conditions, which include MCK1, RIM11, MRK1, and YGK3 that all encode homologues of mammalian glycogen synthase kinase 3 (GSK-3). Ecological analysis of beta diversity between all samples revealed significant population divergence over time and showed feed specific consequences of population structure. Further, we show that significant changes in the population diversity during fed-batch cultivations reflect the presence of significant stresses. Our observations indicate that, for this yeast deletion collection, the selection of the feeding scheme which affects the accumulation of the fermentative by-product ethanol impacts the diversity of the mutant pool to a higher degree as compared to the pH of the culture broth. The mutants that were lost during the time of most extreme population selection suggest that specific biological processes may be required to cope with these specific stresses. CONCLUSIONS: Our results demonstrate the feasibility of Bar-seq to assess fermentation associated stresses in yeast populations under industrial conditions and to understand critical stages of a scale-up process where variability emerges, and selection pressure gets imposed. Overall our work highlights a promising avenue to identify genetic loci and biological stress responses required for fitness under industrial conditions.

Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing.

With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of the organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. The gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes coexist, making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged random barcode transposon sequencing (RB-Tn-Seq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on fatty acids of varying chain lengths indicated specific enzyme substrate preferences and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data, we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with coenzyme A (CoA). Because fatty acids and alcohols may serve as both feedstocks and final products of metabolic-engineering efforts, the fitness data presented here will help guide future genomic modifications toward higher titers, rates, and yields.IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here, we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of the bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and to channel the flux of specific pathway intermediates as desired.

Draft Genome Sequence of Agrobacterium fabrum ARqua1.

Agrobacterium fabrum ARqua1 is a hybrid of Agrobacterium fabrum C58C bearing the megaplasmid pRiA4b. ARqua1 is used by many plant researchers to generate transgenic roots. The draft genome of ARqua1 includes a 249,350-bp contig that likely covers all of pRiA4b, and it will be a valuable resource to plant biologists.

An iron (II) dependent oxygenase performs the last missing step of plant lysine catabolism.

Despite intensive study, plant lysine catabolism beyond the 2-oxoadipate (2OA) intermediate remains unvalidated. Recently we described a missing step in the D-lysine catabolism of Pseudomonas putida in which 2OA is converted to D-2-hydroxyglutarate (2HG) via hydroxyglutarate synthase (HglS), a DUF1338 family protein. Here we solve the structure of HglS to 1.1 Å resolution in substrate-free form and in complex with 2OA. We propose a successive decarboxylation and intramolecular hydroxylation mechanism forming 2HG in a Fe(II)- and O2-dependent manner. Specificity is mediated by a single arginine, highly conserved across most DUF1338 proteins. An Arabidopsis thaliana HglS homolog coexpresses with known lysine catabolism enzymes, and mutants show phenotypes consistent with disrupted lysine catabolism. Structural and biochemical analysis of Oryza sativa homolog FLO7 reveals identical activity to HglS despite low sequence identity. Our results suggest DUF1338-containing enzymes catalyze the same biochemical reaction, exerting the same physiological function across bacteria and eukaryotes.

Chemoinformatic-Guided Engineering of Polyketide Synthases.

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge is re-engineering a partially reductive PKS module to produce a saturated ß-carbon through a reductive loop (RL) exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, offers a viable strategy for RL exchanges. We first introduced a set of donor RLs of diverse genetic origin and chemical substrates  into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with chemical structure similarity between the substrate of the donor RLs and recipient LipPKS1, reaching a titer of 165 mg/L of short-chain fatty acids produced by the host Streptomyces albus J1074. Expanding this method to larger intermediates that require bimodular communication, we introduced RLs of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

Design of orthogonal regulatory systems for modulating gene expression in plants.

Agricultural biotechnology strategies often require the precise regulation of multiple genes to effectively modify complex plant traits. However, most efforts are hindered by a lack of characterized tools that allow for reliable and targeted expression of transgenes. We have successfully engineered a library of synthetic transcriptional regulators that modulate expression strength in planta. By leveraging orthogonal regulatory systems from Saccharomyces spp., we have developed a strategy for the design of synthetic activators, synthetic repressors, and synthetic promoters and have validated their use in Nicotiana benthamiana and Arabidopsis thaliana. This characterization of contributing genetic elements that dictate gene expression represents a foundation for the rational design of refined synthetic regulators. Our findings demonstrate that these tools provide variation in transcriptional output while enabling the concerted expression of multiple genes in a tissue-specific and environmentally responsive manner, providing a basis for generating complex genetic circuits that process endogenous and environmental stimuli.

Osteochondral Lesions of the Talar Dome.

Osteochondral lesion of the talar dome (OCLT) can be a devastating injury that affects mobility. Etiology of these lesions is debated but trauma seems the most supported etiology. Diagnosis of lesions is based on imaging. Conservative management, including weight-bearing restrictions, physical therapy, and supportive measures, often is first-line treatment. Nonsurgical modalities have mixed results and surgical measures often are necessitated for symptom relief. Surgical treatments vary in invasiveness and often are dictated by OCLT size. Studies show patient satisfaction increases substantially after having these procedures performed after failing nonsurgical measures. Results are encouraging, although thorough work-up and discussion should be undertaken.

Leveraging host metabolism for bisdemethoxycurcumin production in Pseudomonas putida.

Pseudomonas putida is a saprophytic bacterium with robust metabolisms and strong solvent tolerance making it an attractive host for metabolic engineering and bioremediation. Due to its diverse carbon metabolisms, its genome encodes an array of proteins and enzymes that can be readily applied to produce valuable products. In this work we sought to identify design principles and bottlenecks in the production of type III polyketide synthase (T3PKS)-derived compounds in P. putida. T3PKS products are widely used as nutraceuticals and medicines and often require aromatic starter units, such as coumaroyl-CoA, which is also an intermediate in the native coumarate catabolic pathway of P. putida. Using a randomly barcoded transposon mutant (RB-TnSeq) library, we assayed gene functions for a large portion of aromatic catabolism, confirmed known pathways, and proposed new annotations for two aromatic transporters. The 1,3,6,8-tetrahydroxynapthalene synthase of Streptomyces coelicolor (RppA), a microbial T3PKS, was then used to rapidly assay growth conditions for increased T3PKS product accumulation. The feruloyl/coumaroyl CoA synthetase (Fcs) of P. putida was used to supply coumaroyl-CoA for the curcuminoid synthase (CUS) of Oryza sativa, a plant T3PKS. We identified that accumulation of coumaroyl-CoA in this pathway results in extended growth lag times in P. putida. Deletion of the second step in coumarate catabolism, the enoyl-CoA hydratase-lyase (Ech), resulted in increased production of the type III polyketide bisdemethoxycurcumin.

Response of Pseudomonas putida to Complex, Aromatic-Rich Fractions from Biomass.

There is strong interest in the valorization of lignin to produce valuable products; however, its structural complexity has been a conversion bottleneck. Chemical pretreatment liberates lignin-derived soluble fractions that may be upgraded by bioconversion. Cholinium ionic liquid pretreatment of sorghum produced soluble, aromatic-rich fractions that were converted by Pseudomonas putida (P. putida), a promising host for aromatic bioconversion. Growth studies and mutational analysis demonstrated that P. putida growth on these fractions was dependent on aromatic monomers but unknown factors also contributed. Proteomic and metabolomic analyses indicated that these unknown factors were amino acids and residual ionic liquid; the oligomeric aromatic fraction derived from lignin was not converted. A cholinium catabolic pathway was identified, and the deletion of the pathway stopped the ability of P. putida to grow on cholinium ionic liquid. This work demonstrates that aromatic-rich fractions obtained through pretreatment contain multiple substrates; conversion strategies should account for this complexity.

Adenosine Triphosphate and Carbon Efficient Route to Second Generation Biofuel Isopentanol.

Climate change necessitates the development of CO2 neutral or negative routes to chemicals currently produced from fossil carbon. In this paper we demonstrate a pathway from the renewable resource glucose to next generation biofuel isopentanol by pairing the isovaleryl-CoA biosynthesis pathway from Myxococcus xanthus and a butyryl-CoA reductase from Clostridium acetobutylicum. The best plasmid and Escherichia coli strain combination makes 80.50 ± 8.08 (SD) mg/L of isopentanol after 36 h under microaerobic conditions with an oleyl alcohol overlay. In addition, the system also shows a strong preference for isopentanol production over prenol in microaerobic conditions. Finally, the pathway requires zero adenosine triphosphate and can be paired theoretically with nonoxidative glycolysis, the combination being redox balanced from glucose thus avoiding unnecessary carbon loss as CO2. These pathway properties make the isovaleryl-CoA pathway an attractive isopentanol production route for further optimization.

Agrobacterium tumefaciens: A Bacterium Primed for Synthetic Biology.

Agrobacterium tumefaciens is an important tool in plant biotechnology due to its natural ability to transfer DNA into the genomes of host plants. Genetic manipulations of A. tumefaciens have yielded considerable advances in increasing transformational efficiency in a number of plant species and cultivars. Moreover, there is overwhelming evidence that modulating the expression of various mediators of A. tumefaciens virulence can lead to more successful plant transformation; thus, the application of synthetic biology to enable targeted engineering of the bacterium may enable new opportunities for advancing plant biotechnology. In this review, we highlight engineering targets in both A. tumefaciens and plant hosts that could be exploited more effectively through precision genetic control to generate high-quality transformation events in a wider range of host plants. We then further discuss the current state of A. tumefaciens and plant engineering with regard to plant transformation and describe how future work may incorporate a rigorous synthetic biology approach to tailor strains of A. tumefaciens used in plant transformation.