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. (C) 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.