Retrons Resolved
by Mechas
Just like solving a crossword puzzle or a sudoku, it is also greatly gratifying when apparently obscure scientific observations are figured out. There are many examples in microbiology of observations that, though they hint at some biological relevance, persistently defy understanding. Perhaps the most recent well-known example is that of CRISPRs (clustered regularly interspaced short palindromic repeats) found in microbial genomes. Years after their identification in bacteria and archaea, CRISPR-Cas systems were found to function in defense against phages and, most noteworthy, have been developed as gene editing tools.
Retrons, which are elements present in numerous bacteria, were identified in the 1980s. They are composed of a non-coding RNA (ncRNA) and a reverse transcriptase (RT) that together generate a covalently linked RNA-DNA hybrid molecule, also known as multi-copy single-stranded DNA (msDNA) (Figure 1). Despite their ubiquity and numerous studies on their possible roles, their biological function had thus far remained elusive, as noted previously in STC.
Figure 1. Retron msDNA Synthesis. (A) Retrons encode reverse transcriptase (RT) and a non-coding RNA (ncRNA) with msr and msd regions. (B) The ncRNA folds to form a stem ending with conserved unpaired guanosine residues on both strands. © The RT uses the 2'-OH of the conserved guanosine as primer to reverse transcribe the msd section which serves as a template, starting from the other conserved guanosine. Degradation with RNase H result in a unique covalently linked RNA-cDNA hybrid called msDNA, which is branched from the guanosine nucleotide (D). Source
In a recent study, Adi Millman and colleagues from Rotem Sorek's lab at the Weizmann Institute show that retrons function as anti-phage defense systems in bacteria. They first searched for genes encoding reverse transcriptases (RT, known to participate in phage defense) that were close to previously reported antiviral defense clusters, such as restriction modification systems. One RT gene was present in various bacterial species and always next to a gene for an endonuclease. When cloned into E. coli MG1655, which lacks such genes, this two-gene system together with the intergenic region provided resistance to phages that belonged to different families. More importantly, this cluster had the hallmark of a retron: an RT gene and a ncRNA precursor in the intergenic region. To verify their observations, the authors made point mutations in regions predicted to affect catalysis of the RT and the reverse transcription of the ncRNA. In both cases mutations abolished anti-phage activity. Strikingly, they found that mutations predicted to affect the activity of the endonuclease also abolished defense against phages. They named this cluster, a three-component defense system, Retron-Eco8.
Figure 2. Multi-Gene Retron Systems. Distribution of different types of genes associated with homologs of retron RTs (N = 4,802). Examples for known retrons are stated below the system type to which they belong. Source
The authors then assessed if retrons in general play a role in defending cells against phages. They identified RT gene homologs located near known defense systems in bacterial and archaeal genomes. These homologs were broadly distributed and consistently associated with additional genes coding for proteins of diverse predicted functions and structures, such as ribosyltransferases or proteins with two predicted transmembrane helices. Thus, the retron functional unit seemed to include associated effector genes that they categorized into 10 types (Figure 2). The authors cloned 11 previously studied retrons from E. coli, Salmonella enterica and Vibrio cholerae into E. coli MG1655 and challenged the resulting strains with phages belonging to different families. Most of these cloned systems provided protection to at least one phage, while some conferred broad defense against phages from different families. Two of these broad range retron systems (Ec48 and Ec73) were studied in greater detail. Point mutations predicted to inactivate the sites crucial for RT and ribosyltransferase activities (in retron Ec73) or that altered the transmembrane helices (retron Ec48) abolished defense, indicating that these associated effector proteins are important for phage defense.
But how do these retron elements prevent viral infection? One clue came from the identification of retron-associated effector genes that code for proteins with domains associated with abortive infection in another anti-phage defense systems, known as CBASS. In abortive infection, infected cells commit suicide to prevent phage replication. Experiments using different multiplicity of infections to infect retron-containing bacteria suggested that these retrons also functioned via abortive infection. Using fluorescence microscopy, the authors then showed that retron Ec48 compromised cell membrane integrity when cells were challenged with λ‑vir phage, causing cells to die and thus limiting viral spread.
Figure 3. Inhibition of RecBCD Triggers Ec48. Model for the anti-phage activity of the Ec48 retron system. Source
To further understand the mechanism involved in defense, the authors identified genes that allowed λ‑vir and T7 phages to escape the Ec48 retron defense system. These genes inhibit the RecBCD bacterial complex, which is central for DNA repair and anti-phage activity. The authors then demonstrated that it is inactivation of RecBCD, and more specifically of RecB, that triggers the Ec48 retron defense system and results in abortive infection (Figure 3).
While many questions remain, this work nicely demonstrates that retrons are composed of a RT, a ncRNA and additional genes for effector proteins that together defend cells from phages. Bacteria and archaea contain numerous and diverse anti-phage defense systems, many of which function via still unidentified mechanisms. The very presence of these elements in microbial genomes hints at the important roles they play in the ongoing host-viral interactions that modulate communities in various niches. Due to their diversity and ubiquity, these defense systems will undoubtedly continue to deliver novel findings regarding the many mechanisms underlying protection against phage infection. More interesting to some will be the possibility of harnessing some of these components for technological applications, as has already been done for example with restriction enzymes and genome editing tools.
