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We grew a soil enrichment culture to identify organisms that anaerobically oxidize phenazine-1-carboxylic acid. A strain of Citrobacter portucalensis was isolated from this enrichment and sequenced by both Illumina and PacBio technologies. It has a genome with a length of 5.3 Mb, a G+C content of 51.8%, and at least one plasmid.
We report the genome of Phormidium yuhuli AB48, which includes a circular chromosome and a circular plasmid (4,747,469 bp and 51,599 bp, respectively). This is currently the only closed reference genome of an isolate of the Phormidium genus, based on the Genome Taxonomy Database (GTDB), providing a potential model system for sustainable biotechnology innovation.
Here, we report the complete genome sequence of the tetrachloroethene-to-trichloroethene dechlorinator Sulfurospirillum sp. strain ACS TCE . The genome consists of a 38.05-kb circular plasmid and a 2.69-Mb circular chromosome, which encodes 3 identical reductive dehalogenases with 91.47% amino acid identity to the PceA of Sulfurospirillum multivorans strain DSM 12446.
We report the complete genome sequence of Acidithiobacillus ferriphilus GT2, an acidophile isolated from gold mill tailings. The circular genome of GT2 contains 2,489 predicted protein-coding units and a single plasmid. Functional analysis indicates the metabolic potential to oxidize iron and reduced sulfur compounds and to fix N 2 and CO 2 .
Sulfurospirillum sp. strain ACS DCE couples growth with reductive dechlorination of tetrachloroethene to cis-1,2-dichloroethene at pH values as low as 5.5. The genome sequence of strain ACS DCE consists of a circular 2,737,849-bp chromosome and a 39,868-bp plasmid and carries 2,737 protein-coding sequences, including two reductive dehalogenase genes.
Antimicrobial resistance in urinary tract infections (UTIs) is a major public health concern. This study aims to characterize the phenotypic and genetic basis of multidrug resistance (MDR) among expanded-spectrum cephalosporin-resistant (ESCR) uropathogenic Escherichia coli (UPEC) causing UTIs in California patient populations. Between February and October 2019, 577 ESCR UPEC isolates were collected from patients at 6 clinical laboratory sites across California. Lineage and antibiotic resistance genes were determined by analysis of whole-genome sequence data. The lineages ST131, ST1193, ST648, and ST69 were predominant, representing 46%, 5.5%, 4.5%, and 4.5% of the collection, respectively. Overall, 527 (91%) isolates had an expanded-spectrum β-lactamase (ESBL) phenotype, with bla CTX-M-15 , bla CTX-M-27 , bla CTX-M-55 , and bla CTX-M-14 being the most prevalent ESBL genes. In the 50 non-ESBL phenotype isolates, 40 (62%) contained bla CMY-2 , which was the predominant plasmid-mediated AmpC (pAmpC) gene. Narrow-spectrum β-lactamases, bla TEM-1B and bla OXA-1 , were also found in 44.9% and 32.1% of isolates, respectively. Among ESCR UPEC isolates, isolates with an ESBL phenotype had a 1.7-times-greater likelihood of being MDR than non-ESBL phenotype isolates (P < 0.001). The cooccurrence of bla CTX-M-15 , bla OXA-1 , and aac(6')-Ib-cr within ESCR UPEC isolates was strongly correlated. Cooccurrence of bla CTX-M-15 , bla OXA-1 , and aac(6')-Ib-cr was associated with an increased risk of nonsusceptibility to piperacillin-tazobactam, cefepime, fluoroquinolones, and amikacin as well as MDR. Multivariate regression revealed the presence of bla CTX-M-55 , bla TEM-1B , and the ST131 genotype as predictors of MDR.
Having the ability to predict the protein-encoding gene content of an incomplete genome or metagenome-assembled genome is important for a variety of bioinformatic tasks. In this study, as a proof of concept, we built machine learning classifiers for predicting variable gene content in Escherichia coli genomes using only the nucleotide k-mers from a set of 100 conserved genes as features. Protein families were used to define orthologs, and a single classifier was built for predicting the presence or absence of each protein family occurring in 10%–90% of all E. coli genomes. The resulting set of 3,259 extreme gradient boosting classifiers had a per-genome average macro F1 score of 0.944 [0.943–0.945, 95% CI]. We show that the F1 scores are stable across multi-locus sequence types and that the trend can be recapitulated by sampling a smaller number of core genes or diverse input genomes. Surprisingly, the presence or absence of poorly annotated proteins, including “hypothetical proteins” was accurately predicted (F1 = 0.902 [0.898–0.906, 95% CI]). Models for proteins with horizontal gene transfer-related functions had slightly lower F1 scores but were still accurate (F1s = 0.895, 0.872, 0.824, and 0.841 for transposon, phage, plasmid, and antimicrobial resistance-related functions, respectively). Finally, using a holdout set of 419 diverse E. coli genomes that were isolated from freshwater environmental sources, we observed an average per-genome F1 score of 0.880 [0.876–0.883, 95% CI], demonstrating the extensibility of the models. Overall, this study provides a framework for predicting variable gene content using a limited amount of input sequence data.
The Web of Registries Search (WoRs) is a web based software application that enables users to search for publicly available biological parts using keywords or sequence fragments. For the initial version (1.0.0) of the application, WoRS targets 10 sources of biological part data: the GenBank NIH genetic sequence database (https://www.ncbi.nlm.nih.gov/genbank/), the iGem parts registry (parts.igem.org), the Addgene plasmid repository (https://www.addgene.org/), and 7 Inventory of Composable Elements (ACS Synbio, JGI, JBEI, JBEI Public, ABF, SynBerc, ABF Public) registry instances. WoRs has built-in automated web scrapers which extract data from sources that do not have a public or well-defined application programming interface (API). They extract as much public data as they can find and create a searchable index to speed up searches. Included in the indexed information is the source of the information.
MVP stands for Multi-choice Viromics Pipeline. It is a pipeline that utilizes a suite of state-of-art tools: geNomad to identify viruses, proviruses, and plasmids in sequencing data, CheckV to assess the quality, and completeness of identified viral genomes, including identification of host contamination for integrated proviruses, A custom code for a rapid genome clustering based on pairwise ANI, Bowtie2, Samtools, and CoverM to calculate coverage of individual viral genomes by read mapping, A custom code to create a vOTU table of abundance, MMseqs2 to compare viral proteins to multiple databases. It provides a quick, and intuitive pipeline to get viral sequences and corresponding properties that can be used for downstream analyses.
Genomad aims to identify mobile genetic elements (namely, viruses and plasmids) from DNA sequence data. It uses a combination of marker gene identification and machine learning models to find likely virus/plasmid candidates in environmental DNA sequencing data. It has an improved classification performance over similar tools.
The Inventory of Composable Elements (ICE) is an open source registry software platform for managing information about biological parts. It is capable of recording information about plasmids, microbial host strains and seeds, as well as DNA parts. Includes features such as DNA sequence visualization, editing and annotation, auto-aligning sequencing trace files against reference templates, SBOL XML/RDF support, and web-of-registries functionality. The web of registries functionality provides strong support for distributed interconnected use and enables sharing and transfer of biological parts across various independent ICE instances. ICE adopts modern software development principles, leveraging component-base frameworks, offering a REST API for convenient third-party integration and emphasizing scalability, security, and service integrations for dynamic content availability. The source code is hosted at https://github.com/JBEI/ice. A public instance is available at public-registry.jbei.org, where users can try out features, upload parts or simply use it for their projects.
The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user’s choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.
Our software program is designed for the Hamilton Vantage liquid handling robot, automating the Build step in the Design-Build-Test-Learn (DBTL) cycle for Saccharomyces cerevisiae. This program minimizes human intervention, enabling rapid identification of pathway bottlenecks and genes that enhance verazine production. The program takes competent yeast and plasmid DNA as input and generates an output library of engineered strains compatible with automated colony picking, high-throughput culturing, and chemical extraction for downstream LC-MS analysis. A user-friendly interface, developed using the Hamilton Method Editor software, allows for on-demand parameter customization. By automating this process, our program streamlines the construction of Saccharomyces cerevisiae, reducing manual labor and increasing efficiency. While the manual process is well-documented, integration with robotic automation is less common, making our program a valuable tool for researchers. With this software, we achieved 2-5 fold increases in verazine production, demonstrating its potential to accelerate research in this field.
Naive and memory CD4 + T cells reactive with human immunodeficiency virus type 1 (HIV-1) are detectable in unexposed, unimmunized individuals. The contribution of preexisting CD4 + T cells to a primary immune response was investigated in 20 HIV-1–seronegative volunteers vaccinated with an HIV-1 envelope (Env) plasmid DNA prime and recombinant modified vaccinia virus Ankara (MVA) boost in the HVTN 106 vaccine trial (clinicaltrials.gov NCT02296541). Prevaccination naive or memory CD4 + T cell responses directed against peptide epitopes in Env were identified in 14 individuals. After priming with DNA, 40% (8/20) of the elicited responses matched epitopes detected in the corresponding preimmunization memory repertoires, and clonotypes were shared before and after vaccination in 2 representative volunteers. In contrast, there were no shared epitope specificities between the preimmunization memory compartment and responses detected after boosting with recombinant MVA expressing a heterologous Env. Preexisting memory CD4 + T cells therefore shape the early immune response to vaccination with a previously unencountered HIV-1 antigen.
Abstract Background Mercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg. Results Our analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg. Conclusions Mer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.
Abstract Background Type I interferons are widely used in research applications and as biotherapeutics. Current assays used to measure interferon concentrations, such as plaque reduction assays and ELISA, are expensive, technically challenging, and may take days to provide results. We sought to develop a robust and rapid assay to determine interferon concentrations produced from transiently transfected cell cultures. Method Indirect quantification of recombinant interferon was evaluated using a novel bi-cistronic construct encoding the Foot-and-mouth disease virus 2A translational interrupter sequence to yield equimolar expression of Gaussia princeps luciferase and porcine interferon α. Direct quantification was evaluated by expression of a novel fusion protein comprised of Gaussia princeps luciferase and porcine type I interferon. Plasmids encoding constructs are transiently transfected into cell cultures and supernatant harvested for testing of luminescence, ELISA determined concentration, and anti-viral activity against vesicular stomatitis virus. Results Bi-cistronic constructs, utilized for indirect quantification, demonstrate both luciferase activity and anti-viral activity. Fusion proteins, utilized for direct quantification, retained secretion and luminescence however only the interferon α fusion protein had antiviral activity comparable to wildtype porcine interferon α. A strong linear correlation was observed between dilution and luminescence for all compounds over a dynamic range of concentrations. Conclusion The correlation of antiviral and luciferase activities demonstrated the utility of this approach, both direct and indirect, to rapidly determine recombinant interferon concentrations. Concentration can be determined over a more dynamic concentration range than available ELISA based assays using this methodology.
Abstract Background Agriculture faces significant global challenges including climate change and an increasing food demand due to a growing population. Addressing these challenges will require the adoption of transformative innovations into biotechnology practice, such as nanotechnology. Recently, nanomaterials have emerged as unmatched tools for their use as biosensors, or as biomolecule delivery vehicles. Despite their increasingly prolific use, plant-nanomaterial interactions remain poorly characterized, drawing into question the breadth of their utility and their broader environmental compatibility. Results Herein, we characterize the response of Arabidopsis thaliana to single walled carbon nanotube (SWNT) exposure with two different surface chemistries commonly used for biosensing and nucleic acid delivery: oligonucleotide adsorbed-pristine SWNTs, and polyethyleneimine-SWNTs loaded with plasmid DNA (PEI-SWNTs), both introduced by leaf infiltration. We observed that pristine SWNTs elicit a mild stress response almost undistinguishable from the infiltration process, indicating that these nanomaterials are well-tolerated by the plant. However, PEI-SWNTs induce a much larger transcriptional reprogramming that involves stress, immunity, and senescence responses. PEI-SWNT-induced transcriptional profile is very similar to that of mutant plants displaying a constitutive immune response or treated with stress-priming agrochemicals. We selected molecular markers from our transcriptomic analysis and identified PEI as the main cause of this adverse reaction. We show that PEI-SWNT response is concentration-dependent and, when persistent over time, leads to cell death. We probed a panel of PEI variant-functionalized SWNTs across two plant species and identified biocompatible SWNT surface functionalizations. Conclusions While SWNTs themselves are well tolerated by plants, SWNTs surface-functionalized with positively charged polymers become toxic and produce cell death. We use molecular markers to identify more biocompatible SWNT formulations. Our results highlight the importance of nanoparticle surface chemistry on their biocompatibility and will facilitate the use of functionalized nanomaterials for agricultural improvement. Graphical Abstract
Abstract Background Zymomonas mobilis is an aerotolerant α-proteobacterium, which has been genetically engineered for industrial purposes for decades. However, a comprehensive comparison of existing strains on the genomic level in conjunction with phenotype analysis has yet to be carried out. We here performed whole-genome comparison of 17 strains including nine that were sequenced in this study. We then compared 15 available Zymomonas strains for their natural abilities to perform under conditions relevant to biofuel synthesis. We tested their growth in anaerobic rich media, as well as growth, ethanol production and xylose utilization in lignocellulosic hydrolysate. We additionally compared their tolerance to isobutanol, flocculation characteristics, and ability to uptake foreign DNA by electroporation and conjugation. Results Using clustering based on 99% average nucleotide identity (ANI), we classified 12 strains into four clusters based on sequence similarity, while five strains did not cluster with any other strain. Strains belonging to the same 99% ANI cluster showed similar performance while significant variation was observed between the clusters. Overall, conjugation and electroporation efficiencies were poor across all strains, which was consistent with our finding of coding potential for several DNA defense mechanisms, such as CRISPR and restriction–modification systems, across all genomes. We found that strain ATCC31821 (ZM4) had a more diverse plasmid profile than other strains, possibly leading to the unique phenotypes observed for this strain. ZM4 also showed the highest growth of any strain in both laboratory media and lignocellulosic hydrolysate and was among the top 3 strains for isobutanol tolerance and electroporation and conjugation efficiency. Conclusions Our findings suggest that strain ZM4 has a unique combination of genetic and phenotypic traits that are beneficial for biofuel production and propose investing future efforts in further engineering of ZM4 for industrial purposes rather than exploring new Zymomonas isolates.