Functional Interactions between Coexisting Toxin-Antitoxin Systems of the ccd Family in Escherichia coli O157:H7

Abstract

Toxin-antitoxin (TA) systems are widely represented on mobile genetic elements as well as in bacterial chromosomes. TA systems encode a toxin and an antitoxin neutralizing it. We have characterized a homolog of the ccd TA system of the F plasmid (ccd F ) located in the chromosomal backbone of the pathogenic O157:H7 Escherichia coli strain (ccd O157 ). The ccd F and the ccd O157 systems coexist in O157:H7 isolates, as these pathogenic strains contain an F-related virulence plasmid carrying the ccd F system. We have shown that the chromosomal ccd O157 system encodes functional toxin and antitoxin proteins that share properties with their plasmidic homologs: the CcdB O157 toxin targets the DNA gyrase, and the CcdA O157 antitoxin is degraded by the Lon protease. The ccd O157 chromosomal system is expressed in its natural context, although promoter activity analyses revealed that its expression is weaker than that of ccd F . ccd O157 is unable to mediate postsegregational killing when cloned in an unstable plasmid, supporting the idea that chromosomal TA systems play a role(s) other than stabilization in bacterial physiology. Our cross-interaction experiments revealed that the chromosomal toxin is neutralized by the plasmidic antitoxin while the plasmidic toxin is not neutralized by the chromosomal antitoxin, whether expressed ectopically or from its natural context. Moreover, the ccd F system is able to mediate postsegregational killing in an E. coli strain harboring the ccd O157 system in its chromosome. This shows that the plasmidic ccd F system is functional in the presence of its chromosomal counterpart.Toxin-antitoxin (TA) proteic systems were originally discovered on low-copy-number plasmids (for reviews on TA systems, see references 11, 22, 24, and 29). They are composed of two genes organized in an operon encoding a toxin and an antitoxin that antagonizes it. The expression of the TA genes is autoregulated at the transcriptional level; the antitoxin acts as a repressor and the toxin often as a corepressor. The antitoxin is an unstable protein degraded by an ATP-dependent protease, while the toxin is a stable protein that inhibits an essential cellular process (e.g., replication and translation). TA systems contribute to plasmid stability by a mechanism called postsegregational killing (PSK). PSK relies on the differential stabilities of the antitoxin and toxin proteins and leads to the killing of daughter bacteria that did not receive a plasmid copy at cell division (31, 50, 53).Recent computational analyses have shown that TA systems are widely represented in eubacterial and archaebacterial chromosomes, suggesting a role for horizontal gene transfer in the spread of these genes (5, 6, 38). The localization of chromosomal TA systems is quite varied. Some are localized within exogenous DNA islands like phages (relBE K-12 in the cryptic lambdoid Qin prophage of Escherichia coli MG1655) (40), transposons (relBE homolog in Tn5401 of Bacillus thuringiensis (23), and superintegrons (relBE, parDE, phd-doc, and hig...

Contribution of the Chromosomal ccdAB Operon to Bacterial Drug Tolerance

“…CcdB F can bind to CcdA O157 but likely with reduced affinity relative to CcdA F , as inferred from the inducer levels at which cells start to die. This is in contrast to a previous study where CcdB F and CcdA O157 were shown to be noninteracting when overexpressed from two different heterologous promoters (32). This is possibly because in the latter case, the CcdA O157 antitoxin is unable to neutralize overexpressed CcdB F completely, and even a small amount of CcdB F can bind to gyrase and cause cell death.…”

“…Homologs of the ccd operon have been discovered on the chromosomes of a variety of bacterial strains, including many pathogenic strains such as those of Shigella dysenteriae, E. coli O157:H7, and Vibrio cholerae. The chromosomal ccd operon cloned on a plasmid shows a lower degree of postsegregational killing than ccdAB F (32). When the ccd operon present on the F plasmid is activated by stress, such as heat or a sublethal dose of antibiotics, an increased frequency of drug-tolerant persisters is observed (20).…”

“…it is expressed alone in the absence of its cognate antidote, CcdA, it causes cell death by poisoning DNA gyrase (32). The ccd operon in this bacterium is located in an intergenic region between two metabolic genes, folA and apaH, which encode dihydrofolate reductase and diadenosine tetraphosphatase, respectively.…”

“…CcdB F , when expressed from the F plasmid, and CcdB O157 , when expressed from the E. coli chromosome, bind to the same target, gyrase A. However, they have been implicated in different functional roles (12,32,33). While the F-plasmid-carried ccd operon is involved in plasmid maintenance by postsegregational killing, no clear role has been attributed to the chromosomal ccd counterpart.…”

“…A previous study has shown that the ccd F system is able to mediate postsegregational killing in an E. coli strain harboring the ccd O157 system on its chromosome. This shows that the plasmid ccd F system is functional even in the presence of its chromosomal counterpart (32). This requires that the CcdA O157 antitoxin either binds weakly or does not bind to the CcdB F toxin.…”