Pan-Chromosome and Comparative Analysis of Agrobacterium fabrum Reveal Important Traits Concerning the Genetic Diversity, Evolutionary Dynamics, and Niche Adaptation of the Species.

Agrobacterium fabrum has been critical for the development of plant genetic engineering and agricultural biotechnology due to its ability to transform eukaryotic cells. However, the gene composition, evolutionary dynamics, and niche adaptation of this species is still unknown. Therefore, we established a comparative genomic analysis based on a pan-chromosome data set to evaluate the genetic diversity of A. fabrum. Here, 25 A. fabrum genomes were selected for analysis by core genome phylogeny combined with the average nucleotide identity (ANI), amino acid identity (AAI), and in silico DNA-DNA hybridization (DDH) values. An open pan-genome of A. fabrum exhibits genetic diversity with variable accessorial genes as evidenced by a consensus pan-genome of 12 representative genomes. The genomic plasticity of A. fabrum is apparent in its putative sequences for mobile genetic elements (MGEs), limited horizontal gene transfer barriers, and potentially horizontally transferred genes. The evolutionary constraints and functional enrichment in the pan-chromosome were measured by the Clusters of Orthologous Groups (COG) categories using eggNOG-mapper software, and the nonsynonymous/synonymous rate ratio (dN/dS) was determined using HYPHY software. Comparative analysis revealed significant differences in the functional enrichment and the degree of purifying selection between the core genome and non-core genome. We demonstrate that the core gene families undergo stronger purifying selection but have a significant bias to contain one or more positively selected sites. Furthermore, although they shared similar genetic diversity, we observed significant differences between chromosome 1 (Chr I) and the chromid in their functional features and evolutionary constraints. We demonstrate that putative genetic elements responsible for plant infection, ecological adaptation, and speciation represent the core genome, highlighting their importance in the adaptation of A. fabrum to plant-related niches. Our pan-chromosome analysis of A. fabrum provides comprehensive insights into the genetic properties, evolutionary patterns, and niche adaptation of the species. IMPORTANCE Agrobacterium spp. live in diverse plant-associated niches such as soil, the rhizosphere, and vegetation, which are challenged by multiple stressors such as diverse energy sources, plant defenses, and microbial competition. They have evolved the ability to utilize diverse resources, escape plant defenses, and defeat competitors. However, the underlying genetic diversity and evolutionary dynamics of Agrobacterium spp. remain unexplored. We examined the phylogeny and pan-genome of A. fabrum to define intraspecies evolutionary relationships. Our results indicate an open pan-genome and numerous MGEs and horizontally transferred genes among A. fabrum genomes, reflecting the flexibility of the chromosomes and the potential for genetic exchange. Furthermore, we observed significant differences in the functional features and evolutionary constraints between the core and accessory genomes and between Chr I and the chromid, respectively.

One-Pot Enzymatic Preparation of Oligonucleotides with an Expanded Genetic Alphabet via Controlled Pause and Restart of Primer Extension: Making Unnatural Out of Natural.

The genetic alphabet of life has been dramatically expanded via the development of unnatural base pairs (UBPs) that work as efficiently as natural base pairs in the storage and retrieval of genetic information. Among the most predominant UBPs, dNaM-dTPT3 and its analogues have been successfully employed to build semisynthetic cells with a functional six-letter genome. With the rapidly growing applications of UBPs in vitro and in vivo, there is an ever-increasing demand for DNA oligonucleotides containing unnatural bases (UBs) at desired positions. Conventional solid-phase synthesis of oligonucleotides has intrinsic limitations and needs to use unstable unnatural phosphoramidites and a DNA synthesizer, so it does not meet the daily urgent requirement for a few UB-containing DNA oligonucleotides in the laboratory. In this work, we develop a one-pot enzymatic method for preparing dNaM- or dTPT3-containing DNA oligonucleotides via controlled pause and restart of primer extension mediated by Klenow fragment (exo-). By systematic optimization of the reaction conditions, high efficiencies and product purities have been achieved. The universality of this method for preparing DNA oligonucleotides containing dNaM or dTPT3 in different sequence contexts is also demonstrated. This method allows convenient production of an arbitrary UB-containing DNA oligonucleotide in a single test tube with only two natural DNA oligonucleotides, stable nucleoside triphosphates, Klenow fragment (exo-), and other common reagents in the laboratory, providing the lowest cost and the highest simplicity for the enzymatic preparation of UB-containing oligonucleotides. Clearly, this method has great potential to facilitate the in vitro and in vivo applications of the UBPs.

Recognition of an Unnatural Base Pair by Tool Enzymes from Bacteriophages and Its Application in the Enzymatic Preparation of DNA with an Expanded Genetic Alphabet.

Unnatural base pairs (UBPs) have been developed to expand the genetic alphabet in vitro and in vivo. UBP dNaM-dTPT3 and its analogues have been successfully used to construct the first set of semi-synthetic organisms, which suggested the great potential of UBPs to be used for producing novel synthetic biological parts. Two prerequisites for doing so are the facile manipulation of DNA containing UBPs with common tool enzymes, including DNA polymerases and ligases, and the easy availability of UBP-containing DNA strands. Besides, for the application of UBPs in phage synthetic biology, the recognition of UBPs by phage enzymes is essential. Here, we first explore the recognition of dNaM-dTPT3 by a family B DNA polymerase from bacteriophage, T4 DNA polymerase D219A. Results from primer extension, steady-state kinetics, and gap-filling experiments suggest that T4 DNA polymerase D219A can efficiently and faithfully replicate dNaM-dTPT3, and efficiently fill a gap by inserting dTPT3TP or its analogues opposite dNaM. We then systematically explore the recognition of dNaM-dTPT3 and its analogues by different DNA ligases from bacteriophages and find that these DNA ligases are generally able to efficiently ligate the DNA nick next to dNaM-dTPT3 or its analogues, albeit with slightly different efficiencies. These results suggest more enzymatic tools for the manipulation of dNaM-dTPT3 and indicate the potential use of dNaM-dTPT3 for expanding the genetic alphabet in bacteriophages. Based on these results, we next develop and comprehensively optimize an upgraded method for enzymatic preparation of unnatural nucleobase (UB)-containing DNA oligonucleotides with good simplicity and universality.

Enzymatic Synthesis of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase.

Development of unnatural base pairs (UBPs) has significantly expanded the genetic alphabet both in vitro and in vivo and led to numerous potential applications in the biotechnology and biopharmaceutical industry. Efficient synthesis of oligonucleotides containing unnatural nucleobases is undoubtedly an essential prerequisite for making full use of the UBPs, and de novo synthesis of oligonucleotides with terminal deoxynucleotidyl transferases (TdTs) has emerged as a method of great potential to overcome limitations of traditional solid-phase synthesis. Herein, we report the efficient template-independent incorporation of nucleotides of unnatural nucleobases dTPT3 and dNaM, which have been designed to make one of the most successful UBPs to date, dTPT3-dNaM, into DNA oligonucleotides with a TdT enzyme under optimized conditions. We also demonstrate the efficient TdT incorporation of dTPT3 derivatives with different functional linkers into oligonucleotides for orthogonal labeling of nucleic acids and applications thereof. The development of a method for the daily laboratory preparation of DNAs with UBPs at arbitrary sites with the assistance of TdT is also described.