Reassembling a cannon in the DNA defense arsenal: genetics of StySA, a BREX phage exclusion system in Salmonella lab strains

Understanding mechanisms that shape horizontal exchange in prokaryotes is a key problem in biology. A major limit on DNA entry is imposed by restriction-modification (RM) processes that depend on the pattern of DNA modification at host-specified sites. In classical RM, endonucleolytic DNA cleavage follows detection of unprotected sites on entering DNA. Recent investigation has uncovered BREX systems, RM-like activities that employ host protection by DNA modification but replication arrest without evident nuclease action on unmodified phage DNA. We show that the historical stySA RM locus of Salmonella enterica sv Typhimurium is a BREX homolog. The stySA29 allele of the hybrid strain LB5000 carries a mutated version of the ancestral LT2 BREX system. Surprisingly, both a restriction and a methylation defect are observed for this lineage despite lack of mutations in brxX, the modification gene homolog. Instead, flanking genes pglZ and brxC each carry multiple mutations (µ) in C-terminal domains. To avoid plasmid artifacts and potential stoichiometric interference, we chose to investigate this system in situ, replacing the mutated pglZµ and brxCµ genes with wild type (WT). PglZ-WT supports methylation in the presence of either BrxCµ or BrxC-WT but not in the presence of a deletion/insertion allele, ΔbrxC::cat. Restriction of phage L requires both BrxC-WT and PglZ-WT, implicating the BrxC C-terminus specifically in restriction activity. Disruption of four other CDS with cat cassettes still permitted modification, suggesting that BrxC, PglZ and BrxX are principal components of the modification activity. BrxL is required for restriction only. A partial disruption of brxL disrupts transcription globally.

Post-Segregational Killing, Altruistic Cell Death, and Other Toxin/Antitoxin Fallacies

The toxin/antitoxin (TA) field, born of controversy, is plagued by several prevailing misperceptions. For example, some TA systems stabilize plasmids and other genomic regions; however, the evidence of post-segregational killing by toxins of TA systems is weak. In addition, there are few credible reports of cell death via TA systems like MazF/MazE and via phage exclusion systems. Although many aspects of the biological roles of TA systems remain enigmatic, there are now some clear, confirmed TA functions: (i) phage inhibition, (ii) plasmid maintenance, (iii) stress response (including regulation of loci distinct from the TA pair itself), (iv) biofilm formation, and (v) persistence. Therefore, this opinion piece aims to challenge the oft-repeated dogma related to TA systems with the goal of emphasizing their primary biological role: constraining metabolism in a reversible manner. Hence, their roles in their five confirmed functions all stem from their ability to rapidly and reversibly reduce metabolic activity.

Characterization of the Major Control Region ofVibrio cholerae Bacteriophage K139: Immunity, Exclusion, and Integration

ABSTRACT The temperate bacteriophage K139 is highly associated with pathogenic O1 Vibrio cholerae strains. The nucleotide sequence of the major control region of K139 was determined. The sequences of four (cox, cII, cI, and int) of the six deduced open reading frames and their gene order indicated that K139 is related to the P2 bacteriophage family. Two genes of the lysogenic transcript from the mapped promoter PL encode homologs to the proteins CI and Int, with deduced functions in prophage formation and maintenance. Between thecI and int genes, two additional genes were identified: orf2, which has no significant similarity to any other gene, and the formerly characterized gene glo. Further analysis revealed that Orf2 is involved in preventing superinfection. In a previous report, we described that mutations inglo cause an attenuation effect in the cholera mouse model (J. Reidl and J. J. Mekalanos, Mol. Microbiol. 18:685–701, 1995). In this report, we present strong evidence that Glo participates in phage exclusion. Glo was characterized to encode a 13.6-kDa periplasmic protein which inhibits phage infection at an early step, hence preventing reinfection of vibriophage K139 into K139 lysogenic cells. Immediately downstream of gene int, the attPsite was identified. Upon analysis of the correspondingattB site within the V. cholerae chromosome, it became evident that phage K139 is integrated between the flagellin genes flaA and flaC of O1 El Tor and O139V. cholerae lysogenic strains.