Transcriptome analyses of cells carrying the Type II Csp231I restriction–modification system reveal cross-talk between two unrelated transcription factors: C protein and the Rac prophage repressor

Abstract

Restriction-modification (R–M) systems represent an effective mechanism of defence against invading bacteriophages, and are widely spread among bacteria and archaea. In acquiring a Type II R–M system via horizontal gene transfer, the new hosts become more resistant to phage infection, through the action of a restriction endonuclease (REase), which recognizes and cleaves specific target DNAs. To protect the host cell's DNA, there is also a methyltransferase (MTase), which prevents DNA cleavage by the cognate REase. In some R–M systems, the host also accepts a cis-acting transcription factor (C protein), which regulates the counteracting activities of REase and MTase to avoid host self-restriction. Our study characterized the unexpected phenotype of Escherichia coli cells, which manifested as extensive cell filamentation triggered by acquiring the Csp231I R–M system from Citrobacter sp. Surprisingly, we found that the cell morphology defect was solely dependent on the C regulator. Our transcriptome analysis supported by in vivo and in vitro assays showed that C protein directly silenced the expression of the RacR repressor to affect the Rac prophage-related genes. The rac locus ydaST genes, when derepressed, exerted a toxicity indicated by cell filamentation through an unknown mechanism. These results provide an apparent example of transcription factor cross-talk, which can have significant consequences for the host, and may represent a constraint on lateral gene transfer.

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

“…Despite the fact the E. coli and Citrobacter are both members of the Family Enterobacteriaceae in the Gammaproteobacteria, Csp231I R-M system expression in E. coli cells without pre-methylation is lethal ( 35 , 52 ). For the Csp231I system to be introduced into E. coli cells, its genome has already to be methylated at the Csp231I target sites ( 60 , 61 ).…”

“…The genetic bases of this phenomenon are not clear. In addition, the effects of C protein gene transfer between two E. coli strains (Table 2 ) might be explained in part by broader regulatory effects of C protein itself ( 52 ).…”

“…Details of their construction is in Supplementary Table S1 , including primers. E. coli DH5α pir was used to propagate pKD-msfGFP ( 50 ), and MG1655 Δ rac ( 51 ) for plasmid fluorescence, R-M system transfer assay and wherever the cross-talk of C protein and transcription factor RacR might be a biological problem ( 52 ). Escherichia coli JM109 served as the host strain for M13 cloning, and MG1655 [F’ ts 114lac::Tn5; Km R ] (gift from Dr George Szatmari, University of Montreal) as the host strain for M13 phage infections.…”

Comparative Genomic Analysis Provides Insights into the Phylogeny, Resistome, Virulome, and Host Adaptation in the Genus Ewingella

“…However, the intact phages in the genomes of the other five E. americana strains could be due to the development of anti-CRISPR systems by phages to avoid CRISPR regulation to enable integration into the genome [47]. It is not surprising to find genes related to R-M systems (type I, II, and IV) among the E. americana strains because R-M systems are widespread and considered as an effective immune system in bacteria and archaea [48,49]. The two to three R-M systems among the strains are consistent with other bacteria.…”

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

“…In a recent report, we studied the TF-controlled Csp231I restriction-modification (R-M) system, during transfer to a new host, in real-time ( 18 , 19 ). The C regulatory protein (TF) precisely controls expression of the two enzymes comprising R-Ms: restriction endonuclease (REase) and DNA methyltransferase (MTase) ( 20–27 ).…”