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We report the construction of an alternative protein priming system to recode genetic translation in Escherichia coli by designing, through trial and error, a chimeric initiator whose sequence identity points partly to elongator tRNA Cys and partly to initiator tRNA f Met . The elaboration of a selection based on the N-terminal cysteine imperative for the function of glucosamine-6-phosphate synthase, an essential enzyme in bacterial cell wall synthesis, was a crucial step to achieve the engineering of this Cys-tRNA j . Iterative improvement of successive versions of Cys-tRNA j was corroborated in vitro by using a biochemical luciferase assay and in vivo by selecting for translation priming of E. coli thymidylate synthase. Condensation assays using specific fluorescent reagent FITC-Gly-cyanobenzothiazole provided biochemical evidence of cysteine coding at the protein priming stage. We showed that translation can be initiated, by N-terminal incorporation of cysteine, at a codon other than UGC by expressing a tRNA j with the corresponding anticodon. The optimized tRNA j is now available to recode the priming of an arbitrary subset of proteins in the bacterial proteome with absolute control of their expression and to evolve the use of xenonucleotides and the emergence of a tXNA j in vivo ., Competing Interests: The authors declare no competing financial interest., (© 2025 The Authors. Published by American Chemical Society.)

Transfer ribonucleic acids (tRNAs) are essential for protein synthesis, decoding mRNA sequences into amino acids. In E. coli K-12 MG1655, 86 tRNA genes are organized in 43 transcription units (TUs) and the essentiality of individual tRNA TUs in bacterial physiology remains unclear. To address this, we systematically generated 43 E. coli tRNA deletion strains in which each tRNA TU was replaced by a kanamycin resistance gene. We found that 33 TUs are not essential for survival, while 10 are essential and require the corresponding TU to be provided on plasmid. The analysis revealed E. coli's tolerance to alterations in tRNA gene copy number and the loss of non-essential tRNAs, as most strains exhibited minimal to no growth differences under various conditions compared to the parental strain. However, deletions metZWV, alaWX and valVW led to significant growth defects under specific conditions. RNA-seq analysis of ∆alaWX and ∆valVW revealed upregulation of genes involved in translation and pilus assembly. Our results provide valuable insights into tRNA dynamics and the cellular response to tRNA TU deletions, paving the way for deeper understanding of tRNA pool complexity., (© 2024. The Author(s).)

We explored the toxicity and mutagenicity of a wide range of xenobiotic nucleoside triphosphates to an Escherichia coli strain equipped with a nucleoside triphosphate transporter. This bacterial test provides a tool to evaluate and guide the synthesis of nucleotides for applications such as the propagation of non-natural genetic information or the selection of potential drugs., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Bacteriophage genomes harbor the broadest chemical diversity of nucleobases across all life forms. Certain DNA viruses that infect hosts as diverse as cyanobacteria, proteobacteria, and actinobacteria exhibit wholesale substitution of aminoadenine for adenine, thereby forming three hydrogen bonds with thymine and violating Watson-Crick pairing rules. Aminoadenine-encoded DNA polymerases, homologous to the Klenow fragment of bacterial DNA polymerase I that includes 3'-exonuclease but lacks 5'-exonuclease, were found to preferentially select for aminoadenine instead of adenine in deoxynucleoside triphosphate incorporation templated by thymine. Polymerase genes occur in synteny with genes for a biosynthesis enzyme that produces aminoadenine deoxynucleotides in a wide array of Siphoviridae bacteriophages. Congruent phylogenetic clustering of the polymerases and biosynthesis enzymes suggests that aminoadenine has propagated in DNA alongside adenine since archaic stages of evolution., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

A synthetic orthogonal polymer embracing a chiral acyclic-phosphonate backbone [( S )-ZNA] is presented that uniquely adds to the emerging family of xenobiotic nucleic acids (XNAs). ( S )-ZNA consists of reiterating six-atom structural units and can be accessed in few synthetic steps from readily available phophonomethylglycerol nucleoside (PMGN) precursors. Comparative thermal stability experiments conducted on homo- and heteroduplexes made of ( S )-ZNA are described that evince its high self-hybridization efficiency in contrast to poor binding of natural complements. Although preliminary and not conclusive, circular dichroism data and dynamic modeling computations provide support to a left-handed geometry of double-stranded ( S )-ZNA. Nonetheless, PMGN diphosphate monomers were recognized as substrates by Escherichia coli ( E. coli) polymerase I as well as being imported into E. coli cells equipped with an algal nucleotide transporter. A further investigation into the in vivo propagation of ( S )-ZNA culminated with the demonstration of the first synthetic nucleic acid with an acyclic backbone that can be transliterated to DNA by the E. coli cellular machinery.

The synthesis, base pairing properties and in vitro (polymerase) and in vivo (E. coli) recognition of 2'-deoxynucleotides with a 2-amino-6-methyl-8-oxo-7,8-dihydro-purine (X), a 2-methyl-6-thiopurine (Y) and a 6-methyl-4-pyrimidone (Z) base moiety are described. As demonstrated by T m measurements, the X and Y bases fail to form a self-complementary base pair. Despite this failure, enzymatic incorporation experiments show that selected DNA polymerases recognize the X nucleotide and incorporate this modified nucleotide versus X in the template. In vivo, X is mainly recognized as a A/G or C base; Y is recognized as a G or C base and Z is mostly recognized as T or C. Replacing functional groups in nucleobases normally involved in W-C recognition (6-carbonyl and 2-amino group of purine; 6-carbonyl of pyrimidine) readily leads to orthogonality (absence of base pairing with natural bases)., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report the design and elaboration of a selection protocol for importing a canonical substrate of DNA polymerase, thymidine triphosphate (dTTP) in Escherichia coli. Bacterial strains whose growth depend on dTTP uptake, through the action of an algal plastid transporter expressed from a synthetic gene inserted in the chromosome, were constructed and shown to withstand the simultaneous loss of thymidylate synthase and thymidine kinase. Such thyA tdk dual deletant strains provide an experimental model of tight nutritional containment for preventing dissemination of microbial GMOs. Our strains transported the four canonical dNTPs, in the following order of preference: dCTP > dATP ≥ dGTP > dTTP. Prolonged cultivation under limitation of exogenous dTTP led to the enhancement of dNTP transport by adaptive evolution. We investigated the uptake of dCTP analogues with altered sugar or nucleobase moieties, which were found to cause a loss of cell viability and an increase of mutant frequency, respectively. E. coli strains equipped with nucleoside triphosphate transporters should be instrumental for evolving organisms whose DNA genome is morphed chemically by fully substituting its canonical nucleotide components.

Although several synthetic or xenobiotic nucleic acids (XNAs) have been shown to be viable genetic materials in vitro, major hurdles remain for their in vivo applications, particularly orthogonality. The availability of XNAs that do not interact with natural nucleic acids and are not affected by natural DNA processing enzymes, as well as specialized XNA processing enzymes that do not interact with natural nucleic acids, is essential. Here, we report 3'-2' phosphonomethyl-threosyl nucleic acid (tPhoNA) as a novel XNA genetic material and a prime candidate for in vivo XNA applications. We established routes for the chemical synthesis of phosphonate nucleic acids and phosphorylated monomeric building blocks, and we demonstrated that DNA duplexes were destabilized upon replacement with tPhoNA. We engineered a novel tPhoNA synthetase enzyme and, with a previously reported XNA reverse transcriptase, demonstrated that tPhoNA is a viable genetic material (with an aggregate error rate of approximately 17 × 10 -3 per base) compatible with the isolation of functional XNAs. In vivo experiments to test tPhoNA orthogonality showed that the E. coli cellular machinery had only very limited potential to access genetic information in tPhoNA. Our work is the first report of a synthetic genetic material modified in both sugar and phosphate backbone moieties and represents a significant advance in biorthogonality toward the introduction of XNA systems in vivo.

The ability of alternative nucleic acids, in which all four nucleobases are substituted, to replicate in vitro and to serve as genetic templates in vivo was evaluated. A nucleotide triphosphate set of 5-chloro-2'-deoxyuridine, 7-deaza-2'-deoxyadenosine, 5-fluoro-2'-deoxycytidine, and 7-deaza-2'deoxyguanosine successfully underwent polymerase chain reaction (PCR) amplification using templates of different lengths (57 or 525mer) and Taq or Vent (exo-) DNA polymerases as catalysts. Furthermore, a fully morphed gene encoding a dihydrofolate reductase was generated by PCR using these fully substituted nucleotides and was shown to transform and confer trimethoprim resistance to E. coli. These results demonstrated that fully modified templates were accurately read by the bacterial replication machinery and provide the first example of a long fully modified DNA molecule being functional in vivo., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

[This corrects the article DOI: 10.1039/C5SC03474D.].

Herein we report the synthesis of N 8 -glycosylated 8-aza-deoxyguanosine ( N 8 -8-aza-dG) and 8-aza-9-deaza-deoxyguanosine ( N 8 -8-aza-9-deaza-dG) nucleotides and their base pairing properties with 5-methyl-isocytosine (d-isoC Me ), 8-amino-deoxyinosine (8-NH 2 -dI), 1- N -methyl-8-amino-deoxyinosine (1-Me-8-NH 2 -dI), 7,8-dihydro-8-oxo-deoxyinosine (8-Oxo-dI), 7,8-dihydro-8-oxo-deoxyadenosine (8-Oxo-dA), and 7,8-dihydro-8-oxo-deoxyguanosine (8-Oxo-dG), in comparison with the d-isoC Me :d-isoG artificial genetic system. As demonstrated by T m measurements, the N 8 -8-aza-dG:d-isoC Me base pair formed less stable duplexes as the C:G and d-isoC Me :d-isoG pairs. Incorporation of 8-NH 2 -dI versus the N 8 -8-aza-dG nucleoside resulted in a greater reduction in T m stability, compared to d-isoC Me :d-isoG. Insertion of the methyl group at the N 1 position of 8-NH 2 -dI did not affect duplex stability with N 8 -8-aza-dG, thus suggesting that the base paring takes place through Hoogsteen base pairing. The cellular interpretation of the nucleosides was studied, whereby a lack of recognition or mispairing of the incorporated nucleotides with the canonical DNA bases indicated the extent of orthogonality in vivo . The most biologically orthogonal nucleosides identified included the 8-amino-deoxyinosines (1-Me-8-NH 2 -dI and 8-NH 2 -dI) and N 8 -8-aza-9-deaza-dG. The 8-oxo modifications mimic oxidative damage ahead of cancer development, and the impact of the MutM mediated recognition of these 8-oxo-deoxynucleosides was studied, finding no significant impact in their in vivo assay.

The synthesis, base-pairing properties and in vitro and in vivo characteristics of 5-methyl-isocytosine (isoC(Me) ) and isoguanine (isoG) nucleosides, incorporated in an HNA(h) (hexitol nucleic acid)-DNA(d) mosaic backbone, are described. The required h-isoG phosphoramidite was prepared by a selective deamination as a key step. As demonstrated by Tm measurements the hexitol sugar showed slightly better mismatch discrimination against dT. The d-isoG base mispairing follows the order T>G>C while the h-isoG base mispairing follows the order G>C>T. The h- and d-isoC(Me) bases mainly mispair with G. Enzymatic incorporation experiments show that the hexitol backbone has a variable effect on selectivity. In the enzymatic assays, isoG misincorporates mainly with T, and isoC(Me) misincorporates mainly with A. Further analysis in vivo confirmed the patterns of base-pair interpretation for the deoxyribose and hexitol isoC(Me) /isoG bases in a cellular context, through incorporation of the bases into plasmidic DNA. Results in vivo demonstrated that mispairing and misincorporation was dependent on the backbone scaffold of the base, which indicates rational advances towards orthogonality., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The templating potential of anhydrohexitol oligonucleotides bearing ambiguous bases was studied in vivo, by using a selection screen for mosaic heteroduplex plasmids in Escherichia coli. 1,5-Anhydro-2,3-dideoxy-2-(5-nitroindazol-1-yl)-D-arabino-hexitol showed the greatest ambiguity among the three nucleosides tested. At most two successive ambiguous bases could be tolerated on hexitol templates read in bacterial cells. Hexitol nucleosides bearing simplified heterocycles thus stand as promising monomers for generating random DNA sequences in vivo from defined synthetic oligonucleotides., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We have constructed a collection of single-gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2-3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.

The C-type lectin DC-SIGN expressed on immature dendritic cells (DCs) captures human immunodeficiency virus (HIV) particles and enhances the infection of CD4+ T cells. This process, known as trans-enhancement of T-cell infection, has been related to HIV endocytosis. It has been proposed that DC-SIGN targets HIV to a nondegradative compartment within DCs and DC-SIGN-expressing cells, allowing incoming virus to persist for several days before infecting target cells. In this study, we provide several lines of evidence suggesting that intracellular storage of intact virions does not contribute to HIV transmission. We show that endocytosis-defective DC-SIGN molecules enhance T-cell infection as efficiently as their wild-type counterparts, indicating that DC-SIGN-mediated HIV internalization is dispensable for trans-enhancement. Furthermore, using immature DCs that are genetically resistant to infection, we demonstrate that several days after viral uptake, HIV transfer from DCs to T cells requires viral fusion and occurs exclusively through DC infection and transmission of newly synthesized viral particles. Importantly, our results suggest that DC-SIGN participates in this process by cooperating with the HIV entry receptors to facilitate cis-infection of immature DCs and subsequent viral transfer to T cells. We suggest that such a mechanism, rather than intracellular storage of incoming virus, accounts for the long-term transfer of HIV to CD4+ T cells and may contribute to the spread of infection by DCs.

Acinetobacter sp. strain ADP1 is a naturally transformable gram-negative bacterium with simple culture requirements, a prototrophic metabolism and a compact genome of 3.7 Mb which has recently been sequenced. Wild-type ADP1 can be genetically manipulated by the direct addition of linear DNA constructs to log-phase cultures. This makes it an ideal organism for the automation of complex strain construction. Here, we demonstrate the flexibility and versatility of ADP1 as a genetic model through the construction of a broad variety of mutants. These include marked and unmarked insertions and deletions, complementary replacements, chromosomal expression tags and complex combinations thereof. In the process of these constructions, we demonstrate that ADP1 can effectively express a wide variety of foreign genes including antibiotic resistance cassettes, essential metabolic genes, negatively selectable catabolic genes and even intact operons from highly divergent bacteria. All of the described mutations were achieved by the same process of splicing PCR, direct transformation of growing cultures and plating on selective media. The simplicity of these tools make genetic analysis and engineering with Acinetobacter ADP1 accessible to laboratories with minimal microbial genetics expertise and very little equipment. They are also compatible with complete automation of genetic analysis and engineering protocols.

HIV-1 Nef protein down-regulates several important immunoreceptors through interactions with components of the intracellular sorting machinery. Nef expression is also known to induce modifications of the endocytic pathway. Here, we analyzed the effects of Nef on retrograde transport, from the plasma membrane to the endoplasmic reticulum using Shiga toxin B-subunit (STxB). Nef expression inhibited access of STxB to the endoplasmic reticulum, but did not modify the surface expression level of STxB receptor, Gb3, nor its internalization rate as measured with a newly developed assay. Mutation of the myristoylation site or of a di-leucine motif of Nef involved in the interaction with the clathrin adaptor complexes AP1 and AP2 abolished the inhibition of retrograde transport. In contrast, mutations of Nef motifs known to interact with PACS-1, beta COP or a subunit of the v-ATPase did not modify the inhibitory activity of Nef on retrograde transport. Ultrastructural analysis revealed that Nef was present in clusters located on endosomal or Golgi membranes together with internalized STxB. Furthermore, in strongly Nef-expressing cells, STxB accumulated in endosomal structures that labeled with AP1. Our observations show that Nef perturbs retrograde transport between the early endosome and the endoplasmic reticulum. The potential transport steps targeted by Nef are discussed.

In principle the hydrogen bonding capacities of 1-(2-deoxy-beta-D-ribofuranosyl)-imidazole-4-carboxamide (dY), and its N-propyl derivative (dYPr), allow them to pair to all four deoxynucleosides. Their triphosphate derivatives (dYTP and dYPrTP) are preferentially incorporated as dATP analogues in a PCR reaction. However, once incorporated into a DNA template their ambiguous hydrogen bonding potential gave rise to misincorporation at frequencies of approximately 3 x 10(-2) per base per amplification. Most of the substitutions were transitions resulting from rotation about the carboxamide bond when part of the template. Between 11-15% of transversions were noted implying rotation of purine or imidazole moieties about the glycosidic bond. As part of a DNA template, dYPr behaved in the same way as dY, despite its propyl moiety. These deoxyimidazole derivatives are among the most radical departures from the canonical bases used so far as substrates in PCR and could be used to generate mutant gene libraries.