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The ability to restrict gene expression to a relevant bacterial species in a complex microbiome is an unsolved problem. In the context of the human microbiome, one desirable target metabolic activity are glucuronide-utilization enzymes (GUS) that are implicated in the toxic re-activation of glucuronidated compounds in the human gastrointestinal (GI) tract, including the chemotherapeutic drug irinotecan. Here, we take advantage of the variable distribution of GUS enzymes in bacteria as a means to distinguish between bacteria with GUS activity, and re-purpose the glucuronide-responsive GusR transcription factor as a biosensor to regulate dCas9 expression in response to glucuronide inducers. We fused the Escherichia coli gusA regulatory region to the dCas9 gene to create pGreg-dCas9, and showed that dCas9 expression is induced by glucuronides, but not other carbon sources. When conjugated from E. coli to Gammaproteobacteria derived from human stool, dCas9 expression from pGreg-dCas9 was restricted to GUS-positive bacteria. dCas9-sgRNAs targeted to gusA specifically down-regulated gus operon transcription in Gammaproteobacteria, with a resulting ∼100-fold decrease in GusA activity. Our data outline a general strategy to re-purpose bacterial transcription factors responsive to exogenous metabolites for precise ligand-dependent expression of genetic tools such as dCas9 in diverse bacterial species.

Metagenomic sequence represents an untapped source of genetic novelty, particularly for conjugative systems that could be used for plasmid-based delivery of Cas9-derived antimicrobial agents. However, unlocking the functional potential of conjugative systems purely from metagenomic sequence requires the identification of suitable candidate systems as starting scaffolds forde novoDNA synthesis. Here, we developed a bioinformatics approach that searches through the metagenomic ‘trash bin’ for conjugative systems present on contigs that are typically excluded from common metagenomic analysis pipelines. Using a human metagenomic gut dataset representing 2805 taxonomically distinct units, we identified 1598 contigs containing conjugative systems with a differential distribution in human cohorts. We synthesizedde novoan entireCitrobacterspp. conjugative system of 54 kb and containing at least 47 genes, pCitro, and found that pCitro conjugates fromEscherichia colitoCitrobacter rodentiumwith a 30-fold higher frequency than toE. coli, and is compatible withCitrobacterresident plasmids. Mutations in thetraVandtraYconjugation components of pCitro inhibited conjugation. We showed that pCitro can be re-purposed as an antimicrobial delivery agent by programming it with the TevCas9 nuclease andCitrobacter-specific sgRNAs to killC. rodentium. Our study reveals a trove of uncharacterized conjugative systems in metagenomic data and describes an experimental framework to animate these large genetic systems as novel target-adapted delivery vectors for Cas9-based editing of bacterial genomes.

D. radiodurans has become an attractive microbial platform for the study of extremophile biology and industrial bioproduction. To improve the genomic manipulation and tractability of this species, the development of tools for whole genome engineering and design is necessary. Here, we report the development of a simple and robust conjugation-based transformation system from E. coli to D. radiodurans allowing for the introduction of stable, replicating plasmids expressing antibiotic resistance markers. Using this method with nonreplicating plasmids, we developed a protocol for creating sequential gene deletions in D. radiodurans by targeting re-striction-modification system genes. Importantly, we demonstrated a conjugation-based method for cloning the large (178 kb), high G+C content MP1 megaplasmid from D. radiodurans in E. coli. The conjugation-based tools described here will facilitate the development of D. radiodurans strains with synthetic genomes for biological studies and industrial applications.Abstract Figure

The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but has also been involved in other cellular functions like telomere regulation and maintenance, in which Ku’s role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells to study the essentiality of Ku70 function. Endogenous Ku70 knockout was achieved using CRISPR/Cas9 editing in cells where Ku70 expression was maintained through integration of an HA-tagged Ku70 cDNA under the control of a doxycycline-inducible promoter. Ku70 conditional knockout cell lines were identified via western blotting, and edits were validated by Sanger sequencing. We visually observed cell death in Ku70 knockout cells 8-10 days post Ku70-HA depletion, and loss of viability following Ku depletion was quantified using crystal violet assays. Interestingly, assessment of telomere length in Ku70 knockout cells using telomere restriction fragment analyses did not reveal any changes in average telomere length following Ku70-HA depletion. Immunofluorescence analysis used to assess γH2AX foci accumulation as a measure of double-stranded DNA breaks following Ku70-HA depletion allowed us to conclude that increased DNA damage is not the driving cause of loss of cell viability. Finally, quantitative proteome analysis of Ku70 knockout cells following Ku70-HA depletion identified a number of pathways and proteins that are significantly dysregulated following the loss of Ku70, including processes which Ku function has been previously associated with such as cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Overall, this conditional Ku70 knockout system reveals that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in other cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.Author SummaryThe Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair, where it acts as a scaffold for other repair factors needed to process double-stranded DNA breaks. Ku has also been involved in other cellular functions like telomere regulation and maintenance, in which Ku’s role is not fully characterized. Previous data suggest that while loss of Ku70/80 can be tolerated in other species, Ku is essential to humans. We have established a conditional Ku70 knockout in HEK293 cells to evaluate the basis of Ku essentiality in human cells. While we observed loss of cell viability upon Ku depletion, we did not observe significant changes in telomere length nor did we record lethal levels of DNA damage upon loss of Ku, suggesting that the reasons for the loss of viability is not linked to the functions of Ku in DNA repair or at telomeres. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study reveals that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.

BackgroundConjugation enables the exchange of genetic elements throughout environments, including the human gut microbiome. Conjugative elements can carry and transfer clinically relevant metabolic pathways which makes precise identification of these systems in metagenomic samples clinically important. ResultsHere, we outline two related methods to identify conjugative elements in the human gut microbiome. Conjugative systems can effectively be identified from metagenomic assemblies either using a curated sets of profile hidden Markov models or by searching against large-scale databases, such as UniRef90. Both methods were successful at identifying type IV conjugative systems with profile hidden Markov models being faster, but less sensitive than alignment to the UniRef database. Finally, we demonstrate that the majority of assembled conjugative elements are not included within metagenomic bins, and that only a small proportion of the binned conjugative systems are included in "high-quality" metagenomic bins.ConclusionsAnalysis of the human gut microbiome by shotgun metagenomic sequencing has revealed numerous connections to human health outcomes. Our findings emphasize the need to identify and analyze conjugative systems outside of standard metagenomic binning pipelines. We suggest that analysis of type IV conjugative systems should be added to the current metagenomic analysis approaches as they contain much information that could explain differences between cohorts.

The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Nanopore long-read sequencing indicates that editing events are characterized by the occurrence of large deletions of up to ~ 2.7 kb centered on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-fluoroorotic acid and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyltransferase activity. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.

The worldwide COVID-19 pandemic caused by the SARS-CoV-2 betacoronavirus has highlighted the need for a synthetic biology approach to create reliable and scalable sources of viral antigen for uses in diagnostics, therapeutics and basic biomedical research. Here, we adapt plasmid-based systems in the eukaryotic microalgae Phaeodactylum tricornutum to develop an inducible overexpression system for SARS-CoV-2 proteins. Limiting phosphate and iron in growth media induced expression of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein from the P. tricornutumHASP1 promoter in the wild-type strain and in a histidine auxotrophic strain that alleviates the requirement for antibiotic selection of expression plasmids. The RBD was purified from whole cell extracts (algae-RBD) with yield compromised by the finding that 90–95% of expressed RBD lacked the genetically encoded C-terminal 6X-histidine tag. Constructs that lacked the TEV protease site between the RBD and C-terminal 6X-histidine tag retained the tag, increasing yield. Purified algae-RBD was found to be N-linked glycosylated by treatment with endoglycosidases, was cross-reactive with anti-RBD polyclonal antibodies, and inhibited binding of recombinant RBD purified from mammalian cell lines to the human ACE2 receptor. We also show that the algae-RBD can be used in a lateral flow assay device to detect SARS-CoV-2 specific IgG antibodies from donor serum at sensitivity equivalent to assays performed with RBD made in mammalian cell lines. Our study shows that P. tricornutum is a scalable system with minimal biocontainment requirements for the inducible production of SARS-CoV-2 or other coronavirus antigens for pandemic diagnostics.

Conjugation enables the exchange of genetic elements throughout environments, including the human gut microbiome. Conjugative elements can carry and transfer clinically relevant metabolic pathways which makes precise identification of these systems in metagenomic samples clinically important. Here, we outline two distinct methods to identify conjugative systems in the human gut microbiome. We first show that conjugative systems exhibit strong population and age-level stratification. Additionally, we find that the total relative abundance of all conjugative systems present in a sample is not an informative metric to use, regardless of the method of identifying the systems. Finally, we demonstrate that the majority of assembled conjugative systems are not included within metagenomic bins, and that only a small proportion of the binned conjugative systems are included in “high-quality” metagenomic bins. Our findings highlight that conjugative systems differ between general North Americans and a cohort of North American pre-term infants, revealing a potential use as an age-related biomarker. Furthermore, conjugative systems can distinguish between other geographical-based cohorts. Our findings emphasize the need to identify and analyze conjugative systems outside of standard metagenomic binning pipelines.ImportanceThe human gut microbiome is increasingly being associated with human health outcomes through shotgun metagenomic sequencing. The usual approach of metagenomic-level analyses is to bin assembled sequences into approximations of bacterial genomes and perform further investigations on the resultant bins. Here, we show that type IV conjugative systems differ between age and geographically-based cohorts and that these systems are systematically excluded by binning algorithms. We suggest that analysis of type IV conjugative systems should be added to the current metagenomic analysis approaches as they contain much information that could explain differences between cohorts beyond those we investigated.

Identifying and validating intermolecular covariation between proteins and their DNA-binding sites can provide insights into mechanisms that regulate selectivity and starting points for engineering new specificity. LAGLIDADG homing endonucleases (meganucleases) can be engineered to bind non-native target sites for gene-editing applications, but not all redesigns successfully reprogram specificity. To gain a global overview of residues that influence meganuclease specificity, we used information theory to identify protein–DNA covariation. Directed evolution experiments of one predicted pair, 227/+3, revealed variants with surprising shifts in I-OnuI substrate preference at the central 4 bases where cleavage occurs. Structural studies showed significant remodeling distant from the covarying position, including restructuring of an inter-hairpin loop, DNA distortions near the scissile phosphates, and new base-specific contacts. Our findings are consistent with a model whereby the functional impacts of covariation can be indirectly propagated to neighboring residues outside of direct contact range, allowing meganucleases to adapt to target site variation and indirectly expand the sequence space accessible for cleavage. We suggest that some engineered meganucleases may have unexpected cleavage profiles that were not rationally incorporated during the design process.

The selective regulation of bacteria in complex microbial populations is key to controlling pathogenic bacteria. CRISPR nucleases can be programmed to kill bacteria, but require an efficient and broad-host range delivery system to be effective. Here, using an Escherichia coli and Salmonella enterica co-culture system, we show that plasmids based on the IncP RK2 conjugative system can be used as delivery vectors for a TevSpCas9 dual nuclease. Notably, a cis-acting plasmid that encodes the conjugation and CRISPR machinery conjugates from E. coli to S. enterica with high frequency compared to a trans system that separates conjugation and CRISPR machinery. In culture conditions that enhance cell-to-cell contact, conjugation rates approach 100% with the cis-acting plasmid. Targeting of single or multiplexed sgRNAs to non-essential genes results in high S. enterica killing efficiencies. Our data highlight the potential of cis-acting conjugative plasmids as a delivery system for CRISPR nucleases or other microbial-altering agents for targeted bacterial killing., CRISPR nucleases can be programmed to cleave sequences in specific bacteria to induce cell death. Here, Hamilton et al. present an optimized method for conjugative delivery of CRISPR nucleases, consisting of a single plasmid that encodes both the conjugative machinery and the nuclease.

Phaeodactylum tricornutum is a marine diatom with a growing genetic toolbox available and is being used in many synthetic biology applications. While most of the genome has been assembled, the currently available genome assembly is not a completed telomere-to-telomere assembly. Here, we used Oxford Nanopore long reads to build a telomere-to-telomere genome for Phaeodactylum tricornutum. We developed a graph-based approach to extract all unique telomeres, and used this information to manually correct assembly errors. In total, we found 25 nuclear chromosomes that comprise all previously assembled fragments, in addition to the chloroplast and mitochondrial genomes. We found that chromosome 19 has filtered long-read coverage and a quality estimate that suggests significantly less haplotype sequence variation than the other chromosomes. This work improves upon the previous genome assembly and provides new opportunities for genetic engineering of this species, including creating designer synthetic chromosomes.

Simple Summary One of the challenges in the emerging field of synthetic biology is engineering organelle genomes. Creating synthetic organelle genomes can open the door to a wide range of applications, such as improving crop yields, treating mitochondrial diseases, or manufacturing high-value chemicals in an environmentally sustainable way. Organelles are tiny biological machines that work inside of living cells. Mitochondria, for example, are responsible for harvesting sugar to create energy for the cell. In previous work, we demonstrated a method to make copies of an alga mitochondrial genome using yeast and bacteria. Algae are of industrial interest for their potential to produce and store large quantities of biofuels and nutritional ingredients. Here, we applied the same approach to copy the mitochondrial genome of a related alga. Although the cloning of this mitochondrial genome in yeast using the previously developed method was possible, the properties of this genome may make it more susceptible to mutations during propagation in bacteria. This work expands our understanding of potential hurdles that can be encountered when cloning and propagating synthetic organelle genomes in host organisms. Abstract Algae are attractive organisms for biotechnology applications such as the production of biofuels, medicines, and other high-value compounds due to their genetic diversity, varied physical characteristics, and metabolic processes. As new species are being domesticated, rapid nuclear and organelle genome engineering methods need to be developed or optimized. To that end, we have previously demonstrated that the mitochondrial genome of microalgae Phaeodactylum tricornutum can be cloned and engineered in Saccharomyces cerevisiae and Escherichia coli. Here, we show that the same approach can be used to clone mitochondrial genomes of another microalga, Thalassiosira pseudonana. We have demonstrated that these genomes can be cloned in S. cerevisiae as easily as those of P. tricornutum, but they are less stable when propagated in E. coli. Specifically, after approximately 60 generations of propagation in E. coli, 17% of cloned T. pseudonana mitochondrial genomes contained deletions compared to 0% of previously cloned P. tricornutum mitochondrial genomes. This genome instability is potentially due to the lower G+C DNA content of T. pseudonana (30%) compared to P. tricornutum (35%). Consequently, the previously established method can be applied to clone T. pseudonana’s mitochondrial genome, however, more frequent analyses of genome integrity will be required following propagation in E. coli prior to use in downstream applications.

The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Editing events are characterized by loss of heterozygosity and by the occurrence of large deletions of up to ~2.7-kb centred on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-FOA and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyl transferase activity in knockout strains. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.

LAGLIDADG homing endonucleases (meganucleases) are site-specific mobile endonucleases that can be adapted for genome-editing applications. However, one problem when reprogramming meganucleases on non-native substrates is indirect readout of DNA shape and flexibility at the central 4 bases where cleavage occurs. To understand how the meganuclease active site regulates DNA cleavage, we used functional selections and deep sequencing to profile the fitness landscape of 1600 I-LtrI and I-OnuI active site variants individually challenged with 67 substrates with central 4 base substitutions. The wild-type active site was not optimal for cleavage on many substrates, including the native I-LtrI and I-OnuI targets. Novel combinations of active site residues not observed in known meganucleases supported activity on substrates poorly cleaved by the wild-type enzymes. Strikingly, combinations of E or D substitutions in the two metal-binding residues greatly influenced cleavage activity, and E184D variants had a broadened cleavage profile. Analyses of I-LtrI E184D and the wild-type proteins co-crystallized with the non-cognate AACC central 4 sequence revealed structural differences that correlated with kinetic constants for cleavage of individual DNA strands. Optimizing meganuclease active sites to enhance cleavage of non-native central 4 target sites is a straightforward addition to engineering workflows that will expand genome-editing applications.

With the completion of the genome sequence, and development of an efficient conjugation-based transformation system allowing the introduction of stable episomes, Phaeodactylum tricornutum has become an ideal platform for the study of diatom biology and synthetic biology applications. The development of plasmid-based genetic tools is the next step to improve manipulation of this species. Here, we report the identification of endogenous P. tricornutum promoters and terminators allowing selective expression of antibiotic resistance markers from stably replicating plasmids in P. tricornutum. Significantly, we developed a protocol for sequential conjugation of plasmids from Escherichia coli to P. tricornutum and demonstrated simultaneous replication of two plasmids in P. tricornutum. We developed a simple and robust conjugative system for Cas9 editing that yielded up to 60% editing efficiency of the urease gene. Finally, we constructed a plasmid encoding eight genes involved in vanillin biosynthesis that was propagated in P. tricornutum over four months with no evidence of rearrangements, with whole-plasmid sequencing indicating that the majority of mutations occurred after plasmid assembly and initial conjugation rather than during long-term propagation. The plasmid-based tools described here will facilitate investigation of the basic biology of P. tricornutum and enable synthetic biology applications.

Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth. Conjugation within solid media could also provide a more stable microenvironment in which the conjugative pilus can establish and maintain contact with recipient cells for the successful delivery of plasmid DNA. Furthermore, transfer in solid media may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp. Using our optimized method, plasmids of varying sizes were tested for transfer from Escherichia coli to Saccharomyces cerevisiae. We demonstrated that there was no significant change in conjugation frequency when plasmid size increased from 56.5 to 138.6 kbp in length. Finally, we established an efficient PCR-based synthesis protocol to generate custom conjugative plasmids.

Pathogenic mitochondrial DNA (mtDNA) mutations often co‐exist with wild‐type molecules (mtDNA heteroplasmy). Phenotypes manifest when the percentage of mutant mtDNA is high (70–90%). Previously, our laboratory showed that mitochondria‐targeted transcription activator‐like effector nucleases (mitoTALENs) can eliminate mutant mtDNA from heteroplasmic cells. However, mitoTALENs are dimeric and relatively large, making it difficult to package their coding genes into viral vectors, limiting their clinical application. The smaller monomeric GIY‐YIG homing nuclease from T4 phage (I‐TevI) provides a potential alternative. We tested whether molecular hybrids (mitoTev‐TALEs) could specifically bind and cleave mtDNA of patient‐derived cybrids harboring different levels of the m.8344A>G mtDNA point mutation, associated with myoclonic epilepsy with ragged‐red fibers (MERRF). We tested two mitoTev‐TALE designs, one of which robustly shifted the mtDNA ratio toward the wild type. When this mitoTev‐TALE was tested in a clone with high levels of the MERRF mutation (91% mutant), the shift in heteroplasmy resulted in an improvement of oxidative phosphorylation function. mitoTev‐TALE provides an effective architecture for mtDNA editing that could facilitate therapeutic delivery of mtDNA editing enzymes to affected tissues.

Diatoms are an ecologically important group of eukaryotic microalgae with properties that make them attractive for biotechnological applications such as biofuels, foods, cosmetics and pharmaceuticals. Phaeodactylum tricornutum is a model diatom with defined culture conditions, but routine genetic manipulations are hindered by a lack of simple and robust genetic tools. One obstacle to efficient engineering of P. tricornutum is that the current selection methods for P. tricornutum transformants depend on the use of a limited number of antibiotic resistance genes. An alternative and more cost-effective selection method would be to generate auxotrophic strains of P. tricornutum by knocking out key genes involved in amino acid biosynthesis, and using plasmid-based copies of the biosynthetic genes as selective markers. Previous work on gene knockouts in P. tricornutum used biolistic transformation to deliver CRISPR-Cas9 system into P. tricornutum. Biolistic transformation of non-replicating plasmids can cause undesired damage to P. tricornutum due to random integration of the transformed DNA into the genome. Subsequent curing of edited cells to prevent long-term overexpression of Cas9 is very difficult as there is currently no method to excise integrated plasmids. This protocol adapts a new method to deliver the Cas9 or TevCas9 system into P. tricornutum via conjugation of plasmids from a bacterial donor cell. The process involves: 1) design and insertion of a guideRNA targeting the P. tricornutum urease gene into a TevCas9 expression plasmid that also encodes a conjugative origin of transfer, 2) installation of this plasmid in Escherichia coli containing a plasmid (pTA-Mob) containing the conjugative machinery, 3) transfer of the TevCas9 expression plasmid into P. tricornutum by conjugation, 4) screening of ex-conjugants for urease knockouts using T7 Endonuclease I and phenotypic screening, and 5) curing of the plasmid from edited cells.

Breakthroughs in the development of programmable site-specific nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases (MNs), and most recently, the clustered regularly interspaced short palindromic repeats (CRISPR) associated proteins (including Cas9) have greatly enabled and accelerated genome editing. By targeting double-strand breaks to user-defined locations, the rates of DNA repair events are greatly enhanced relative to un-catalyzed events at the same sites. However, the underlying biology of each genome-editing nuclease influences the targeting potential, the spectrum of off-target cleavages, the ease-of-use, and the types of recombination events at targeted double-strand breaks. No single genome-editing nuclease is optimized for all possible applications. Here, we focus on the diversity of nuclease domains available for genome editing, highlighting biochemical properties and the potential applications that are best suited to each domain.

Staphylococcus aureus requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for S. aureus physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed S. aureus uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that S. aureus can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a trans-acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a cis-acting leucine-responsive attenuator, uncovering how S. aureus regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in S. aureus, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on S. aureus environmental adaptation and virulence via CodY., Author summary To infect its human host, the bacterial pathogen Staphylococcus aureus must either take up nutrients from the surrounding environment or produce them itself. Previous studies have reported that S. aureus does not produce the amino acids leucine and valine, despite it possessing the genes to do so. In this study, we show that S. aureus does indeed produce leucine and valine, but only under certain nutritional conditions. We select for mutants of S. aureus able to grow in valine-depleted environments to uncover genetic variants that enable valine production. We discover genetic variants in a repressor protein and a region of non-coding DNA that both, when properly functioning, inhibit production of leucine and valine under nutrient-rich conditions. We further identify the nutritional conditions where the inhibition of leucine and valine production is relieved, thus revealing a previously overlooked role for another amino acid, isoleucine, in influencing nutrient metabolism. We show that isoleucine levels also influence expression of genes involved in the ability of S. aureus to cause disease. These findings help to reconcile conflicting reports regarding leucine and valine production in S. aureus and reveal nutritional cues that could influence its ability to cause infection.

Precise genome editing in complex genomes is enabled by engineered nucleases that can be programmed to cleave in a site-specific manner. Here, we fused the small, sequence-tolerant monomeric nuclease domain from the homing endonuclease I-TevI to transcription-like activator effectors (TALEs) to create monomeric Tev-TALE nucleases (Tev-mTALENs). Using the PthXo1 TALE scaffold to optimize the Tev-mTALEN architecture, we found that choice of the N-terminal fusion point on the TALE greatly influenced activity in yeast-based assays, and that the length of the linker used affected the optimal spacing of the TALE binding site from the I-TevI cleavage site, specified by the motif 5′-CNNNG-3′. By assaying activity on all 64 possible sequence variants of this motif, we discovered that in the Tev-mTALEN context, I-TevI prefers A/T-rich triplets over G/C-rich ones at the cleavage site. Profiling of nucleotide requirements in the DNA spacer that separates the CNNNG motif from the TALE binding site revealed substantial, but not complete, tolerance to sequence variation. Tev-mTALENs showed robust mutagenic activity on an episomal target in HEK 293T cells consistent with specific cleavage followed by nonhomologous end-joining repair. Our data substantiate the applicability of Tev-mTALENs as genome-editing tools but highlight DNA spacer and cleavage site nucleotide preferences that, while enhancing specificity, do confer moderate targeting constraints.

The CRISPR/Cas9 nuclease is commonly used to make gene knockouts. The blunt DNA ends generated by cleavage can be efficiently ligated by the classical nonhomologous end-joining repair pathway (c-NHEJ), regenerating the target site. This repair creates a cycle of cleavage, ligation, and target site regeneration that persists until sufficient modification of the DNA break by alternative NHEJ prevents further Cas9 cutting, generating a heterogeneous population of insertions and deletions typical of gene knockouts. Here, we develop a strategy to escape this cycle and bias events toward defined length deletions by creating an RNA-guided dual active site nuclease that generates two noncompatible DNA breaks at a target site, effectively deleting the majority of the target site such that it cannot be regenerated. The TevCas9 nuclease, a fusion of the I-TevI nuclease domain to Cas9, functions robustly in HEK293 cells and generates 33- to 36-bp deletions at frequencies up to 40%. Deep sequencing revealed minimal processing of TevCas9 products, consistent with protection of the DNA ends from exonucleolytic degradation and repair by the c-NHEJ pathway. Directed evolution experiments identified I-TevI variants with broadened targeting range, making TevCas9 an easy-to-use reagent. Our results highlight how the sequence-tolerant cleavage properties of the I-TevI homing endonuclease can be harnessed to enhance Cas9 applications, circumventing the cleavage and ligation cycle and biasing genome-editing events toward defined length deletions.

Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth. Conjugation within solid media could also provide a more stable microenvironment in which the conjugative pilus can establish and maintain contact with recipient cells for the successful delivery of plasmid DNA. Furthermore, transfer in solid media may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp. Using our optimized method, plasmids of varying sizes were tested for transfer from Escherichia coli to Saccharomyces cerevisiae. We demonstrated that there was no significant change in conjugation frequency when plasmid size increased from 56.5 to 138.6 kbp in length. Finally, we established an efficient PCR-based synthesis protocol to generate custom conjugative plasmids.

The GIY-YIG nuclease domain is found within protein scaffolds that participate in diverse cellular pathways and contains a single active site that hydrolyzes DNA by a one-metal ion mechanism. GIY-YIG homing endonucleases (GIY-HEs) are two-domain proteins with N-terminal GIY-YIG nuclease domains connected to C-terminal DNA-binding and they are thought to function as monomers. Using I-BmoI as a model GIY-HE, we test mechanisms by which the single active site is used to generate a double-strand break. We show that I-BmoI is partially disordered in the absence of substrate, and that the GIY-YIG domain alone has weak affinity for DNA. Significantly, we show that I-BmoI functions as a monomer at all steps of the reaction pathway and does not transiently dimerize or use sequential transesterification reactions to cleave substrate. Our results are consistent with the I-BmoI DNA-binding domain acting as a molecular anchor to tether the GIY-YIG domain to substrate, permitting rotation of the GIY-YIG domain to sequentially nick each DNA strand. These data highlight the mechanistic differences between monomeric GIY-HEs and dimeric or tetrameric GIY-YIG restriction enzymes, and they have implications for the use of the GIY-YIG domain in genome-editing applications.

Targeted manipulation of complex genomes often requires the introduction of a double-strand break at defined locations by site-specific DNA endonucleases. Here, we describe a monomeric nuclease domain derived from GIY-YIG homing endonucleases for genome-editing applications. Fusion of the GIY-YIG nuclease domain to three-member zinc-finger DNA binding domains generated chimeric GIY-zinc finger endonucleases (GIY-ZFEs). Significantly, the I-TevI-derived fusions (Tev-ZFEs) function in vitro as monomers to introduce a double-strand break, and discriminate in vitro and in bacterial and yeast assays against substrates lacking a preferred 5′-CNNNG-3′ cleavage motif. The Tev-ZFEs function to induce recombination in a yeast-based assay with activity on par with a homodimeric Zif268 zinc-finger nuclease. We also fused the I-TevI nuclease domain to a catalytically inactive LADGLIDADG homing endonuclease (LHE) scaffold. The monomeric Tev-LHEs are active in vivo and similarly discriminate against substrates lacking the 5′-CNNNG-3′ motif. The monomeric Tev-ZFEs and Tev-LHEs are distinct from the FokI-derived zinc-finger nuclease and TAL effector nuclease platforms as the GIY-YIG domain alleviates the requirement to design two nuclease fusions to target a given sequence, highlighting the diversity of nuclease domains with distinctive biochemical properties suitable for genome-editing applications.

Summary The td group I intron interrupting the thymidylate synthase (TS) gene of phage T4 is a mobile intron that encodes the homing endonuclease I-TevI. Efficient RNA splicing of the intron is required to restore function of the TS gene, while expression of I-TevI from within the intron is required to initiate intron mobility. Three distinct layers of regulation temporally limit I-TevI expression to late in the T4 infective cycle, yet the biological rationale for stringent regulation has not been tested. Here, we deleted key control elements to deregulate I-TevI expression at early and middle times post T4 infection. Strikingly, we found that deregulation of I-TevI, or of a catalytically inactive variant, generated a thymidine-dependent phenotype that is caused by a reduction in td intron splicing. Prematurely terminating I-TevI translation restores td splicing, full-length TS synthesis, and rescues the thymidine-dependent phenotype. We suggest that stringent translational control of I-TevI evolved to prevent the ribosome from disrupting key structural elements of the td intron that are required for splicing and TS function at early and middle times post T4 infection. Analogous translational regulatory mechanisms in unrelated intron-open reading frame arrangements may also function to limit deleterious consequences on splicing and host gene function.

Insight into protein structure and function is best obtained through a synthesis of experimental, structural and bioinformatic data. Here, we outline a framework that we call MUSE (mutual information, unigenic evolution and structure-guided elucidation), which facilitated the identification of previously unknown residues that are relevant for function of the GIY-YIG homing endonuclease I-BmoI. Our approach synthesizes three types of data: mutual information analyses that identify co-evolving residues within the GIY-YIG catalytic domain; a unigenic evolution strategy that identifies hyper- and hypo-mutable residues of I-BmoI; and interpretation of the unigenic and co-evolution data using a homology model. In particular, we identify novel positions within the GIY-YIG domain as functionally important. Proof-of-principle experiments implicate the non-conserved I71 as functionally relevant, with an I71N mutant accumulating a nicked cleavage intermediate. Moreover, many additional positions within the catalytic, linker and C-terminal domains of I-BmoI were implicated as important for function. Our results represent a platform on which to pursue future studies of I-BmoI and other GIY-YIG-containing proteins, and demonstrate that MUSE can successfully identify novel functionally critical residues that would be ignored in a traditional structure-function analysis within an extensively studied small domain of approximately 90 amino acids.

Homing endonucleases are site-specific DNA endonucleases that typically function as mobile genetic elements by introducing a double-strand break (DSB) in genomes that lack the endonuclease, resulting in a unidirectional gene conversion event that mobilizes the homing endonuclease gene and flanking DNA. Here, we characterize phage T4-encoded mobE, a predicted free-standing HNH family homing endonuclease. We show that mobE is promoterless and dependent on upstream transcription for expression, and that an internal intrinsic terminator regulates mobE transcript levels. Crucially, in vivo mapping experiments revealed a MobE-dependent, strand-specific nick in the non-coding strand of the nrdB gene of phage T2. An internal deletion of the predicted HNH catalytic motif of MobE abolishes nicking, and reduces high-frequency inheritance of mobE. Sequence polymorphisms of progeny phage that inherit mobE are consistent with DSB repair pathways. Significantly, we found that mobility of the neighboring I-TevIII, a defunct homing endonuclease encoded within a group I intron interrupting the nrdB gene of phage T4, was dependent on an intact mobE gene. Thus, our data indicate that the stagnant nrdB intron and I-TevIII are mobilized in trans as a consequence of a MobE-dependent gene conversion event, facilitating persistence of genetic elements that have no inherent means of promoting their own mobility.

Previous transcript mapping of the bacteriophage Aeh1 nrd operon revealed a predicted RNA hairpin upstream of the homing endonuclease mobE gene. We enzymatically mapped the hairpin, showing that the mobE ribosome binding site is sequestered. Cloning of the hairpin upstream of lacZ resulted in reduced β-galactosidase activity, consistent with translational regulation.

Mobile genetic elements have the potential to influence the expression of genes surrounding their insertion site upon invasion of a genome. Here, we examine the transcriptional organization of a ribonucleotide reductase operon ( nrd ) that has been invaded by an HNH family homing endonuclease, mobE . In Aeromonas hydrophila phage Aeh1, mobE has inserted into the large-subunit gene ( nrdA ) of aerobic ribonucleotide reductase (RNR), splitting it into two smaller genes, nrdA-a and nrdA-b . This gene organization differs from that in phages T4, T6, RB2, RB3, RB15, and LZ7, where mobE is inserted in the nrdA-nrdB intergenic region. We present evidence that the expression of Aeh1 mobE is regulated by transcriptional, posttranscriptional, and translational controls. An Aeh1-specific late promoter drives expression of mobE , but strikingly the mobE transcript is processed internally at an RNase E-like site. We also identified a putative stem-loop structure upstream of mobE that sequesters the mobE ribosome binding site, presumably acting to down regulate MobE translation. Moreover, our transcriptional analyses indicate that the surrounding nrd genes of phage Aeh1 are expressed by a different strategy than are the corresponding phage T4 genes and that transcriptional readthrough is the only mechanism by which the promoterless Aeh1 nrdB gene is expressed. We suggest that the occurrence of multiple layers of control to limit the expression of mobE to late in the Aeh1 infection cycle is an adaptation of Aeh1 to reduce any effects on expression of the surrounding nrd genes early in phage infection when RNR function is critical.

Staphylococcus aureus can utilize ferric hydroxamates as a source of iron under iron-restricted growth conditions. Proteins involved in this transport process are: FhuCBG, which encodes a traffic ATPase; FhuD2, a post-translationally modified lipoprotein that acts as a high affinity receptor at the cytoplasmic membrane for the efficient capture of ferric hydroxamates; and FhuD1, a protein with similarity to FhuD2. Gene duplication likely gave rise to fhuD1 and fhuD2. While the genomic locations of fhuCBG and fhuD2 in S. aureus strains are conserved, both the presence and the location of fhuD1 are variable. The apparent redundancy of FhuD1 led us to examine the role of this protein. We demonstrate that FhuD1 is expressed only under conditions of iron limitation through the regulatory activity of Fur. FhuD1 fractions with the cell membrane and binds hydroxamate siderophores but with lower affinity than FhuD2. Using small angle x-ray scattering, the solution structure of FhuD1 resembles that of FhuD2, and only a small conformational change is associated with ferrichrome binding. FhuD1, therefore, appears to be a receptor for ferric hydroxamates, like FhuD2. Our data to date suggest, however, that FhuD1 is redundant to FhuD2 and plays a minor role in hydroxamate transport. However, given the very real possibility that we have not yet identified the proper conditions where FhuD1 does provide an advantage over FhuD2, we anticipate that FhuD1 serves an enhanced role in the transport of untested hydroxamate siderophores and that it may play a prominent role during the growth of S. aureus in its natural environments.

Customary binding sites of intron-encoded homing endonucleases lie within cognate intronless alleles, at the so-called homing sites. Here, we describe a novel, high-affinity binding site for I-TevI endonuclease, encoded within the group I td intron of phage T4. This site is an operator that overlaps the T4 late promoter, which drives I-TevI expression from within the td intron. I-TevI binds the operator and homing sites with equal affinity, and functions as a transcriptional autorepressor. Distinct sequence and spacing requirements of the catalytic domain result in reduced cleavage activity on operator DNA. Crystallographic studies showed that the overall interactions of the DNA-binding domain with the operator and homing sites are similar, but have some different hydrogen-bonding contacts. We present a model in which the flexibility in protein-DNA interactions allows I-TevI to bind variant intronless alleles to promote intron mobility while facilitating its function in autorepression, and thereby persistence in its host.

Homing endonucleases initiate mobility of their host group I introns by binding to and cleaving lengthy recognition sequences that are typically centered on the intron insertion site (IS) of intronless alleles [1, 2]. Because the intron interrupts the endonucleases' recognition sequence, intron-containing alleles are immune to cleavage by their own endonuclease [3]. I- Tev I and I- Bmo I are related GIY-YIG endonucleases that bind a homologous stretch of thymidylate synthase (TS)-encoding DNA but use different strategies to distinguish intronless from intron-containing substrates [4–8]. I- Tev I discriminates between substrates at the level of DNA binding, as its recognition sequence is centered on the intron IS [5–7]. I- Bmo I, in contrast, possesses a very asymmetric recognition sequence with respect to the intron IS, binds both intron-containing and intronless TS-encoding substrates, but efficiently cleaves only intronless substrate [8]. Here, we show that I- Bmo I is extremely tolerant of multiple substitutions around its cleavage sites and has a low specific activity. However, a single G-C base pair, at position −2 of a 39-base pair recognition sequence, is a major determinant for cleavage efficiency and distinguishes intronless from intron-containing alleles. Strikingly, this G-C base pair is universally conserved in phylogenetically diverse TS-coding sequences; this finding suggests that I- Bmo I has evolved exquisite cleavage requirements to maximize the potential to spread to variant intronless alleles, while minimizing cleavage at its own intron-containing allele.

Targeting gene disruptions in complex genomes relies on imprecise repair by the non-homologous end-joining DNA pathway, creating mutagenic insertions or deletions (indels) at the break point. DNA end-processing enzymes are often co-expressed with genome-editing nucleases to enhance the frequency of indels, as the compatible cohesive ends generated by the nucleases can be precisely repaired, leading to a cycle of cleavage and non-mutagenic repair. Here, we present an alternative strategy to bias repair toward gene disruption by fusing two different nuclease active sites from I-TevI (a GIY-YIG enzyme) and I-OnuI E2 (an engineered meganuclease) into a single polypeptide chain. In vitro, the MegaTev enzyme generates two double-strand breaks to excise an intervening 30-bp fragment. In HEK 293 cells, we observe a high frequency of gene disruption without co-expression of DNA end-processing enzymes. Deep sequencing of disrupted target sites revealed minimal processing, consistent with the MegaTev sequestering the double-strand breaks from the DNA repair machinery. Off-target profiling revealed no detectable cleavage at sites where the I-TevI CNNNG cleavage motif is not appropriately spaced from the I-OnuI binding site. The MegaTev enzyme represents a small, programmable nuclease platform for extremely specific genome-engineering applications.

The active sites of enzymes consist of residues necessary for catalysis and structurally important noncatalytic residues that together maintain the architecture and function of the active site. Examples of evolutionary interactions between catalytic and noncatalytic residues have been difficult to define and experimentally validate due to a general intolerance of these residues to substitution. Here, using computational methods to predict coevolving residues, we identify a network of positions consisting of two catalytic metal-binding residues and two adjacent noncatalytic residues in LAGLIDADG homing endonucleases (LHEs). Distinct combinations of the four residues in the network map to distinct LHE subfamilies, with a striking distribution of the metal-binding Asp (D) and Glu (E) residues. Mutation of these four positions in three LHEs--I-LtrI, I-OnuI, and I-HjeMI--indicate that the combinations of residues tolerated are specific to each enzyme. Kinetic analyses under single-turnover conditions revealed that I-LtrI activity could be modulated over an ∼100-fold range by mutation of residues in the coevolving network. I-LtrI catalytic site variants with low activity could be rescued by compensatory mutations at adjacent noncatalytic sites that restore an optimal coevolving network and vice versa. Our results demonstrate that LHE activity is constrained by an evolutionary barrier of residues with strong context-dependent effects. Creation of optimal coevolving active-site networks is therefore an important consideration in engineering of LHEs and other enzymes.

Group I introns are intervening sequences that have invaded tRNA, rRNA and protein coding genes in bacteria and their phages. The ability of group I introns to self-splice from their host transcripts, by acting as ribozymes, potentially renders their insertion into genes phenotypically neutral. Some group I introns are mobile genetic elements due to encoded homing endonuclease genes that function in DNA-based mobility pathways to promote spread to intronless alleles. Group I introns have a limited distribution among bacteria and the current assumption is that they are benign selfish elements, although some introns and homing endonucleases are a source of genetic novelty as they have been co-opted by host genomes to provide regulatory functions. Questions regarding the origin and maintenance of group I introns among the bacteria and phages are also addressed.

Homing endonucleases and other site-specific endonucleases have potential applications in genome editing, yet efficient targeting requires a thorough understanding of DNA-sequence specificity. Here, we describe a modified two-plasmid genetic selection in Escherichia coli that allows rapid profiling of nucleotide substitutions within a target site of given endonucleases. The selection utilizes a toxic plasmid (pTox) that encodes a DNA gyrase toxin in addition to the endonuclease target site. Cleavage of the toxic plasmid by an endonuclease expressed from a second plasmid (pEndo) facilitates growth under selective conditions. The modified protocol utilizes competent cells harboring the endonuclease expression plasmid into which target site plasmids are transformed. Replica plating on nonselective and selective media plates identifies cleavable and non-cleavable targets. Thus, a library of randomized target sites, or many individual target sites, can be analyzed using a single transformation. Both cleavable and non-cleavable targets can be analyzed by DNA sequencing to gain information about nucleotide preference in the endonuclease's target site.