Regulator-dependent temporal dynamics of a restriction-modification system's gene expression upon entering new host cells: single-cell and population studies

Abstract

Restriction-modification (R-M) systems represent a first line of defense against invasive DNAs, such as bacteriophage DNAs, and are widespread among bacteria and archaea. By acquiring a Type II R-M system via horizontal gene transfer, the new hosts generally become more resistant to phage infection, through the action of a restriction endonuclease (REase), which cleaves DNA at or near specific sequences. A modification methyltransferase (MTase) serves to protect the host genome against its cognate REase activity. The production of R-M system components upon entering a new host cell must be finely tuned to confer protective methylation before the REase acts, to avoid host genome damage. Some type II R-M systems rely on a third component, the controller (C) protein, which is a transcription factor that regulates the production of REase and/or MTase. Previous studies have suggested C protein effects on the dynamics of expression of an R-M system during its establishment in a new host cell. Here, we directly examine these effects. By fluorescently labelling REase and MTase, we demonstrate that lack of a C protein reduces the delay of REase production, to the point of being simultaneous with, or even preceding, production of the MTase. Single molecule tracking suggests that a REase and a MTase employ different strategies for their target search within host cells, with the MTase spending much more time diffusing in proximity to the nucleoid than does the REase. This difference may partially ameliorate the toxic effects of premature REase expression.

Visual evidence for the recruitment of four enzymes with RNase activity to the Bacillus subtilis replication forks

“…These data show that like DNA transcription factors (Stracy et al, 2021), or sequencespecific DNA methylases (Negri et al, 2021), H-type RNases and ExoR mostly employ constrained motion through the nucleoids, diffusing between DNA strands to find binding targets, which would explain the rather low mobility of the high-mobile fraction.…”

“…Heat maps of all single molecule trajectories showed a clear enrichment of tracks at central places, even within nucleoid areas, similar to those of DNA polymerase C, and very different from the more peripheral pattern of RNase J1, which is involved in RNA degradation within the membrane-associated RNA degradosome. These data show that like DNA transcription factors (Stracy et al ., 2021), or sequence-specific DNA methylases (Negri et al ., 2021), H-type RNases and ExoR mostly employ constrained motion through the nucleoids, diffusing between DNA strands to find binding targets, which would explain the rather low mobility of the high-mobile fraction.…”

Molecular basis for lethal cross-talk between two unrelated bacterial transcription factors - the regulatory protein of a restriction-modification system and the repressor of a defective prophage

“…The C protein gene was fused to the mVenus fluorescent protein gene, and tested for use in SMT technology ( Supplementary Figures S4 and S5 of Supplementary Materials). The SMT setup we used is explained in ( 28 , 42 ). Briefly, the central part of a 514 nm laser beam was used for stream acquisition (20 ms integration time) of mVenus fusions, and movies were captured by a Hamamatsu ImageEM EMCCD camera (128×128 pixel area of chip used).…”

“…The C regulatory protein (TF) precisely controls expression of the two enzymes comprising R-Ms: restriction endonuclease (REase) and DNA methyltransferase (MTase) ( 20–27 ). This is especially crucial during the operon transfer to a new host, when C protein acts to delay REase expression so the MTase has time to completely modify the genome to prevent its damage due to REase cutting ( 28–30 ). Even though the Csp231I R-M system is located on the chromosome, when placed on a plasmid it disseminates easily within the Enterobacteriaceae members which naturally exchange their genetic material via conjugation/mobilization and transduction ( 28 , 31 ).…”

“…This is especially crucial during the operon transfer to a new host, when C protein acts to delay REase expression so the MTase has time to completely modify the genome to prevent its damage due to REase cutting ( 28–30 ). Even though the Csp231I R-M system is located on the chromosome, when placed on a plasmid it disseminates easily within the Enterobacteriaceae members which naturally exchange their genetic material via conjugation/mobilization and transduction ( 28 , 31 ). Using a combination of genetics and transcriptomics we observed that, due to this TF’s action, the host's genetic network expression was changed, affecting cell fitness, as manifested by cell filamentation (Figure 1 ).…”