Group II Intron Protein Localization and Insertion Sites Are Affected by Polyphosphate

Abstract

Mobile group II introns consist of a catalytic intron RNA and an intron-encoded protein with reverse transcriptase activity, which act together in a ribonucleoprotein particle to promote DNA integration during intron mobility. Previously, we found that the Lactococcus lactis Ll.LtrB intron-encoded protein (LtrA) expressed alone or with the intron RNA to form ribonucleoprotein particles localizes to bacterial cellular poles, potentially accounting for the intron's preferential insertion in the oriC and ter regions of the Escherichia coli chromosome. Here, by using cell microarrays and automated fluorescence microscopy to screen a transposon-insertion library, we identified five E. coli genes (gppA, uhpT, wcaK, ynbC, and zntR) whose disruption results in both an increased proportion of cells with more diffuse LtrA localization and a more uniform genomic distribution of Ll.LtrB-insertion sites. Surprisingly, we find that a common factor affecting LtrA localization in these and other disruptants is the accumulation of intracellular polyphosphate, which appears to bind LtrA and other basic proteins and delocalize them away from the poles. Our findings show that the intracellular localization of a group II intron-encoded protein is a major determinant of insertion-site preference. More generally, our results suggest that polyphosphate accumulation may provide a means of localizing proteins to different sites of action during cellular stress or entry into stationary phase, with potentially wide physiological consequences.

The Take and Give Between Retrotransposable Elements and their Hosts

“…Suppression suggests that the effect of ppGpp is mediated via RNA polymerase. Although not all ppGpp mutants depress retromobility, possibly due to the different host strains and assay systems used, the above studies provide a satisfying correspondence of regulatory effects mediated via phosphate metabolism and suggest that nutritional stress may be an activator of group II intron mobility (48,210).…”

“…Small molecules: polyphosphates, (p)ppGpp and cAMP-Recent work has identified five genes in E. coli, gppA, uhpT, wcaK, ynbC, and zntR, whose disruption leads to a more diffuse cellular distribution of the IEP, accompanied by a more uniform distribution of intron integrations around the genome (210). The common factor in these mutants is accumulation of intracellular polyphosphate, which was also shown to bind to LtrA and to alter distribution of other pole-localized basic proteins.…”

Group II Introns: Mobile Ribozymes that Invade DNA

“…As expected, pathways using nascent DNA strands as primers are favored under rapid growth conditions, which lead to an increased frequency of replication forks (Coros et al 2005). Retromobility of the Ll.LtrB intron is also influenced in interesting ways by cellular interactions, host factors, and stress responses (Beauregard et al 2008;Zhao et al 2008;Coros et al 2009). …”

Mobile Bacterial Group II Introns at the Crux of Eukaryotic Evolution

“…These studies also gave the first hint that culture conditions can regulate retrotransposition, as slow growth eliminated all events except those around ori and ter and confined them to an endonucleasedependent pathway into dsDNA (59). Additionally, a mutagenesis screen showed that Ll.LtrB IEP localization is related to its interaction with negatively charged intracellular polyphosphates, which are normally polelocalized but delocalize away from the poles in response to cellular stress, leading to a more uniform distribution of intron-insertion sites (96). These studies portended growth phase and cellular stress as regulatory forces in retrotransposition.…”