UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions

Abstract

Reverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs. In this work, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential.

Figure 1.

Phylogenetic tree of 5022 representative UG/Abi RT sequences. Alignment of the RT domains 1–7 was used as an input for FastTree (Methods). For visualization, midpoint rooting was applied using FigTree, and clades were collapsed to the UG/Abi group level according to the presence of reference sequences showing a FastTree support > 0.70 (Supplementary File S1) with UFBoot and SH-aLRT support values in the IQ-TREE (not shown) >90%. Due to the presence of reference sequence in two groups, UG13 UG/Abi group was subdivided (UG13a and UG13b) according to phylogenetic criteria and protein length distribution. Numbers in brackets and segments sizes represent the absolute number of representative sequences in each group. Branches corresponding o UG/Abi Classes 1, 2 and 3 are colored in brick red, turquoise, and olive, respectively. The FastTree phylogenetic tree and Multiple Sequence Alignment of the RT domain can be found in NEXUS format as Supplementary File S1.

Figure 2.

Graphical summary and classification of the various UG/Abi RT systems. Genes are represented as arrows of size proportional to their length. For clarity, the UG3 + UG8 system is included within Class 1, but notice that the UG3 RT is phylogenetically placed within Class 2. The identified domains are represented in different colors. The NCBI Protein and Nucleotide accession IDs of chosen representative sequences are shown to the left. Numbers inside genes indicate the cluster to which they belong, and the names above indicate domain annotation. Genes with dotted lines represent genes that are occasionally present. Vertical dashed lines are represented to be 600 nucleotides (200 aminoacids) apart. Shadows between genes of different variants indicate similar domains. Icons on the left of each group's name represent those that have been experimentally validated as defense systems and those that are considered to be novel. Predicted ncRNAs are indicated by dots with differentiated colors according to the group.

Figure 3.

(A) Multiple structural alignment of predicted protein structures of Class 1 UG/Abi RTs C-terminal regions visualized using using cealign feature from Pymol and visualized using PyMol2Rainbow rainbow spectrum. (B) Heatmap of the pairwise TM-scores obtained using the mTM-align algorithm. The file containing the multiple structural alignment can be found as Supplementary File S3.

Figure 4.

(A) Phage plaque assays showing resistance of E. coli harboring plasmids with different UG/Abi RTs against coliphages T2, T5 and ZL-19. Variants with mutations in the catalytic domains of RT and/or effector proteins, as well as deletions of the C-terminal αRep in UG15 are also included. RT, reverse transcriptase; PrimS, primase. (B) Predicted consensus structure of ncRNAs found in UG28 systems.

Figure 5.

Gene-sharing network of UG27-containing viral genomes dereplicated at 95% and similar viruses found in reference databases (Materials and Methods). Nodes represent viral genomes and edges connect genomes with similar gene content. Edge opacity is proportional to vContact2 weights which represent the significance of the relationships. Node colors indicate the family to which every genome belongs based on the APS-tree, and circle color shades around nodes represent the four major families found. Nodes with no connections to UG27-containing nodes were removed to improve the visualization.