Insertion of a homing endonuclease creates a genes-in-pieces ribonucleotide reductase that retains function

Abstract

In bacterial and phage genomes, coding regions are sometimes interrupted by self-splicing introns or inteins, which can encode mobility-promoting homing endonucleases. Homing endonuclease genes are also found free-standing (not intron-or intein-encoded) in phage genomes where they are inserted in intergenic regions. One example is the HNH family endonuclease, mobE, inserted between the large (nrdA) and small (nrdB) subunit genes of aerobic ribonucleotide reductase (RNR) of T-even phages T4, RB2, RB3, RB15, and LZ7. Here, we describe an insertion of mobE into the nrdA gene of Aeromonas hydrophila phage Aeh1. The insertion creates a unique genes-in-pieces arrangement, where nrdA is split into two independent genes, nrdA-a and nrdA-b, each encoding cysteine residues that correspond to the active-site residues of uninterrupted NrdA proteins. Remarkably, the mobE insertion does not inactivate NrdA function, although the insertion is not a self-splicing intron or intein. We copurified the NrdA-a, NrdA-b, and NrdB proteins as complex from Aeh1-infected cells and also showed that a reconstituted complex has RNR activity. Class I RNR activity in phage Aeh1 is thus assembled from separate proteins that interact to form a composite active site, demonstrating that the mobE insertion is phenotypically neutral in that its presence as an intervening sequence does not disrupt the function of the surrounding gene.bacteriophage Aeh1 ͉ gene structure ͉ intervening sequence H oming endonucleases are a distinctive class of site-specific DNA endonucleases that promote the lateral transfer of their own coding region and flanking DNA between genomes by a recombination-dependent process termed homing (reviewed in ref. 1). Homing endonucleases are often encoded within self-splicing introns and inteins (2-4), but many bacterial and phage genomes possess a significant number of so-called freestanding homing endonucleases that are not encoded within introns or inteins (5, 6). Free-standing endonucleases do not have the benefit of a self-splicing element to minimize their impact on host gene structure and function and, thus, are found at genomic insertion sites that are of low impact, such as intergenic regions. In T4-like phages that infect Escherichia coli and related bacteria, free-standing endonucleases are more abundant than intron-encoded versions (6-9) and promote their spread to phage genomes lacking the endonuclease by an intronless homing pathway (10-12).As a consequence of their abundance in T4-like phages, free-standing endonucleases represent a significant source of genetic variation by promoting recombination between genomes. Many characterized homing endonucleases have recognition sites that lie in genes that function in DNA metabolism, including those that encode aerobic and anaerobic ribonucleotide reductases (RNRs) (13). Three classes of RNRs have been described to date, based on required metallocofactors used to generate a radical intermediate and on structural differences (14). Prokaryotic class Ia aerobic enzymes, typifi...

Genomic and Proteomic Characterization of the Broad-Host-Range Salmonella Phage PVP-SE1: Creation of a New Phage Genus

“…Promoters were screened using the string search engine in Kodon for the consensus sequence (Ϫ35)TTGACAN [15][16][17][18] TATAAT(Ϫ10), with only one potential error. Potential rho-independent terminators were identified using MFOLD (83).…”

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“…pertaining to phages, and not bacteria), including the large subunit of terminases, portal proteins, and structural proteins. Other phage-specific ORFs, which are frequently found in annotated viral genomes, such as those belonging to the cro or cI-repressor system (important in lytic and lysogenic development (Folkmanis et al 1977)) and homing HNH endonucleases (important in lateral transfer (Friedrich et al 2007)), were also identified in the Metavir hits, albeit far fewer in number (1315 and 3855 respectively). This suggests the presence of lysogenic or potentially lysogenic phages in the Lake Michigan viromes.…”

A homing endonuclease and the 50-nt ribosomal bypass sequence of phage T4 constitute a mobile DNA cassette

“…In one case, a GIY-YIG family endonuclease gene disrupts DNA polymerase (28,29); in the other case, a H-N-H family endonuclease gene disrupts the large subunit of ribonucleotide reductase (30). In all three cases, the products of the split genes reunite to form enzymatically active proteins.…”