The decay of the chromosomally encoded ccdO157 toxin-antitoxin system in the Escherichia coli species - PubMed (original) (raw)

The decay of the chromosomally encoded ccdO157 toxin-antitoxin system in the Escherichia coli species

Natacha Mine et al. Genetics. 2009 Apr.

Abstract

The origin and the evolution of toxin-antitoxin (TA) systems remain to be uncovered. TA systems are abundant in bacterial chromosomes and are thought to be part of the flexible genome that originates from horizontal gene transfer. To gain insight into TA system evolution, we analyzed the distribution of the chromosomally encoded ccdO157 system in 395 natural isolates of Escherichia coli. It was discovered in the E. coli O157:H7 strain in which it constitutes a genomic islet between two core genes (folA and apaH). Our study revealed that the folA-apaH intergenic region is plastic and subject to insertion of foreign DNA. It could be composed (i) of a repetitive extragenic palindromic (REP) sequence, (ii) of the ccdO157 system or subtle variants of it, (iii) of a large DNA piece that contained a ccdAO157 antitoxin remnant in association with ORFs of unknown function, or (iv) of a variant of it containing an insertion sequence in the ccdAO157 remnant. Sequence analysis and functional tests of the ccdO157 variants revealed that 69% of the variants were composed of an active toxin and antitoxin, 29% were composed of an active antitoxin and an inactive toxin, and in 2% of the cases both ORFs were inactive. Molecular evolution analysis showed that ccdBO157 is under neutral evolution, suggesting that this system is devoid of any biological role in the E. coli species.

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Figures

F<sc>igure</sc> 1.—

Figure 1.—

PCR detection of the ccdO157 system in the E. coli species. The ccdO157 system is located between the folA and the apaH gene (not on scale). Its presence was detected by PCR using the P40 and P93 primers that are complementary to the ORFs that are flanking the ccdO157 system. Another set of primers (P43 and P47) complementary to the ccdAO157 and ccdBO157 ORFs was used to detect it at other potential chromosomal locations in strains that did not carry ccdO157 between folA and apaH.

F<sc>igure</sc> 2.—

Figure 2.—

Diversity of the folA–apaH region in the E. coli species. PCR reactions were carried out on E. coli isolates using P40 and P93. Lane 1, molecular weight marker (SMART ladder, Eurogentec), size is indicated in base pairs; lane 2, MG1655; lane 3, O157 (2886-75, STEC Center); lane 4, O153 (LREC); lane 5, O163 (LREC); lane 6, O86 (LREC); lane 7, negative control. The size (base pairs) of the different amplicons is indicated.

F<sc>igure</sc> 3.—

Figure 3.—

Diversity of the folA–apaH region within the O153 serogroup. PCR reactions were carried out on three O153 isolates using P40 and P93. Lane 1, molecular weight marker (SMART ladder, Eurogentec), size is indicated in base pairs; lane 2, negative control; lane 3, HC45; lane 4, MG1655; lane 5, HC71; lane 6, O153 (LREC). The size (base pairs) of the different amplicons is indicated.

F<sc>igure</sc> 4.—

Figure 4.—

Plasticity of the folA–apaH IR within the E. coli species. (A) Representation of the diversity of the folA–apaH IR. The strains and the size (base pairs) of the different folA–apaH intergenic regions (IR) are indicated. The folA and apaH genes are represented in black. The ccdAO157 and ccdBO157 genes are represented in white and dark gray, respectively (indicated as A and B). A′ represents the 3′-terminal part of the ccdAO157 gene. ORF2 (129 amino acids) is represented in light gray, ORF3 (199 amino acids) in white, and IS_621_ in stippled white. (B) Sequence alignment of the seq1 sequence and CcdAO157. The amino acid sequences were aligned with CLUSTALW. Symbols: asterisk, identical amino acids; colon, strongly similar amino acids; period, weakly similar amino acids. The total number of amino acids for each protein is in parentheses.

F<sc>igure</sc> 5.—

Figure 5.—

Properties of the ccdO51 and ccdO138 variants. (A) The CcdAO138 protein is not able to counteract the toxicity of CcdBO157. SG22622/pKK223-3/pBAD33-ccdBO157 (triangle), SG22622/pKK-ccdAO138/pBAD33-ccdBO157 (square), and SG22622/pKK-ccdAO157/pBAD33-ccdBO157 (diamond) were grown in the presence of 1% arabinose. (B) The CcdBO51 and CcdBO138 variants are not toxic. SG22622/pBAD33 (diamond), SG22622/pBAD33-ccdBO51 (square), SG22622/pBAD33-ccdBO138 (circle) and SG22622/pBAD33-ccdBO157 (triangle) were grown in the presence of 1% arabinose. In A and B serial dilutions of the cultures were plated at regular time intervals on CCM plates without arabinose and incubated overnight at 37°. Values correspond to the mean of results of three independent experiments and SDs are indicated. (C) The CcdAO51 protein is expressed in E. coli O51 and counteracts the toxicity of CcdBO157 expressed in trans. O51 (triangle) and O51Δ_ccdO51_ (square) containing the pBAD33 vector (solid symbols) or the pBAD33-ccdBO157 plasmid (open symbols) were grown in the presence of 0.25% arabinose. At regular time intervals, serial dilutions of the cultures were plated on CCM plates without arabinose and incubated overnight at 37°. Values correspond to the mean of results of three independent experiments and SDs are indicated.

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