The hokW-sokW Locus Encodes a Type I Toxin–Antitoxin System That Facilitates the Release of Lysogenic Sp5 Phage in Enterohemorrhagic Escherichia coli O157

The toxin-antitoxin (TA) genetic modules control various bacterial events, such as plasmid maintenance, persister cell formation, and phage defense. They also exist in mobile genetic elements, including prophages; however, their physiological roles remain poorly understood. Here, we demonstrate that hokW-sokW, a putative TA locus encoded in Sakai prophage 5 (Sp5) in enterohemorrhagic Escherichia coli O157: H7 Sakai strain, functions as a type I TA system. Bacterial growth assays showed that the antitoxic activity of sokW RNA against HokW toxin partially requires an endoribonuclease, RNase III, and an RNA chaperone, Hfq. We also demonstrated that hokW-sokW assists Sp5-mediated lysis of E. coli cells when prophage induction is promoted by the DNA-damaging agent mitomycin C (MMC). We found that MMC treatment diminished sokW RNA and increased both the expression level and inner membrane localization of HokW in a RecA-dependent manner. Remarkably, the number of released Sp5 phages decreased by half in the absence of hokW-sokW. These results suggest that hokW-sokW plays a novel role as a TA system that facilitates the release of Sp5 phage progeny through E. coli lysis.

The effect of type II toxin-antitoxin systems on methicillinresistant Staphylococcus aureus persister cell formation and antibiotic tolerance

Persister cells are defi ned as a subpopulation of bacteria in a dormant state with the ability to reduce bacterial metabolism and they are involved in antibiotic tolerance. Toxin-antitoxin (TA) systems have been previously suggested as important players in persistence. Therefore, this study aimed to study the involvement of TA systems in persister cell formation in methicillin-resistant Staphylococcus aureus following antibiotic exposure. Using TADB and RASTA database, two type II TA systems including MazF/MazE and RelE/RelB were predicted in S. aureus. The presence of these TA genes was determined in 5 methicillin-resistant S. aureus isolates and the standard strain S. aureus subsp. aureus N315 using PCR method. To induce persistence, isolates were exposed to lethal doses of ciprofl oxacin and the expression of the studied TA system genes was measured after 5 h using Real-Time PCR. According to our results, all the studied isolates harbored the TA system genes. S. aureus was highly capable of persister cell formation following exposure to sub-MIC of ciprofl oxacin and RT-qPCR showed a signifi cant increase in the expression of the MazEF and RelBE loci, indicating their potential role in antibiotic tolerance. Considering the importance of antibiotic tolerance, further studies on persister cell formation and TA systems involved in this phenomenon are required to effi ciently target these systems.

CRISPR-Cas Controls Cryptic Prophages

The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be non-functional in the best-studied bacterium, Escherichia coli K-12. Instead, we demonstrate here that the E. coli CRISPR-Cas system is active and inhibits its nine defective (i.e., cryptic) prophages. Specifically, deactivation of CRISPR-Cas via deletion of cas2, which encodes one of the two conserved CRISPR-Cas proteins, reduces growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation; hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages. Consistently, seven of the 13 E. coli spacers contain matches to the cryptic prophages, and, after excision, CRISPR-Cas cleaves cryptic prophage CP4-57 and DLP-12 DNA. Moreover, we determine that the key genes in these cryptic prophages that CRISPR-Cas represses by cleaving the excised DNA include lysis protein YdfD of Qin and lysis protein RzoD of DLP-12. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages; i.e., unlike with active lysogens, CRISPR-Cas and cryptic prophages may stably exist.

Escherichia coli Cryptic Prophages Sense Nutrients to Control Persister Cell Resuscitation

We determined previously that some cryptic prophages are not genomic junk but instead enable cells to combat myriad stresses as part of an active stress response. However, how these phage fossils affect the extreme stress response of dormancy; i.e., how cryptic prophages affect persister cell formation and resuscitation, has not been fully explored. Persister cells form as a result of stresses such as starvation, antibiotics, and oxidative conditions, and resuscitation of these persister cells likely causes recurring infections such as those associated with tuberculosis, cystic fibrosis, and Lyme disease. Unlike for the active stress response, here we find that deletion of each of the nine Escherichia coli cryptic prophages has no effect on persister cell formation. Strikingly, elimination of each cryptic prophage results in an increase in persister cell resuscitation with a dramatic increase in resuscitation upon deleting all nine prophages. This increased resuscitation includes eliminating the need for a carbon source and is due to activation of the phosphate import system as a result of inactivating transcriptional regulator AlpA of the CP4-57 cryptic prophage, since we found alpA increases persister resuscitation, and AlpA represses phosphate regulator PhoR. Therefore, we report a novel cellular stress mechanism controlled by cryptic prophages: regulation of phosphate uptake which controls the exit of the cell from dormancy and prevents premature resuscitation in the absence of nutrients.

Biofilm Persister Cell Formation, and Relative Gene Expression Analysis of Type II Toxin-Antitoxin System in Pseudomonas Aeruginosa Strains in the Exponential and Stationary Phases

Chronic and persistent infections and therapy failure are concerning issues in patients with Pseudomonas aeruginosa infections. Presence of persister cells in biofilm considers as one from the causes for antibiotic resistance and treatment failure. Consequently, in this research, the expression of TA type II systems into persister formation of biofilm was assessed in presence of colistin ciprofloxacin antibiotics into exponential and stationary phases. The results showed that TA systems at different stages of bacterial growth in biofilm have different functions that subsequently, cause differences in the presence amount of persister cells at each stage of bacterial growth. The expression of the systems were measured by Real-Time PCR method. Keywords: Persister cell; Biofilm; Pseudomonas aeruginosa; TA systems; Real-time PCR; Exponential and Stationary phases

The Expression of Type II TA System Genes Following Persister Cell Formation in Pseudomonas Aeruginosa Isolates in The Exponential and Stationary Phases

Objectives: Failure of infection therapy in the presence of antibiotics has become a major problem which has been mostly attributed to the ability of bacterial persister cell formation. Bacteria use various mechanisms to form persister cells in different phases, among which is the toxin-antitoxin (TA) systems. This study aimed at investigating the expression of type II TA system genes under the stress of ciprofloxacin and colistin antibiotics in the exponential and stationary phases.Methods: To determine the effects of ciprofloxacin and colistin on persister cell formation in the exponential and stationary phases of Pseudomonas aeruginosa strains, colony counting was performed at different time intervals in the presence of 5-fold MIC of ciprofloxacin and colistin. In addition, the expression of relBE, Xre-COG5654, vapBC, and Xre-GNAT genes in P. aeruginosa isolates was assessed 3.5 h after antibiotic treatment in the exponential and stationary phases using qRT-PCR.Results: Our results indicated the presence of persister phenotype of P. aeruginosa strains in the presence of 5-fold MIC of ciprofloxacin and colistin compared to the control after 3.5 h of incubation in the exponential and stationary phases. Also, the number of persister cells in the stationary phase was higher than that of the exponential phase. According to the results of qRT-PCR, ciprofloxacin and colistin may induce persister cells by increasing the expression of type II TA systems in stationary and exponential phases.Conclusion: Ciprofloxacin and colistin may increase the formation of persister cells by affecting the expression of type II TA systems.