Spontaneous excision of the Salmonella enterica serovar Enteritidis-specific defective prophage-like element phiSE14

Abstract

Salmonella enterica serovar Enteritidis has emerged as a major health problem worldwide in the last few decades. DNA loci unique to S. Enteritidis can provide markers for detection of this pathogen and may reveal pathogenic mechanisms restricted to this serovar. An in silico comparison of 16 Salmonella genomic sequences revealed the presence of an approximately 12.5-kb genomic island (GEI) specific to the sequenced S. Enteritidis strain NCTC13349. The GEI is inserted at the 5' end of gene ydaO (SEN1377), is flanked by 308-bp imperfect direct repeats (attL and attR), and includes 21 open reading frames (SEN1378 to SEN1398), encoding primarily phage-related proteins. Accordingly, this GEI has been annotated as the defective prophage SE14 in the genome of strain NCTC13349. The genetic structure and location of phiSE14 are conserved in 99 of 103 wild-type strains of S. Enteritidis studied here, including reference strains NCTC13349 and LK5. Notably, an extrachromosomal circular form of phiSE14 was detected in every strain carrying this island. The presence of attP sites in the circular forms detected in NCTC13349 and LK5 was confirmed. In addition, we observed spontaneous loss of a tetRA-tagged version of phiSE14, leaving an empty attB site in the genome of strain NCTC13349. Collectively, these results demonstrate that phiSE14 is an unstable genetic element that undergoes spontaneous excision under standard growth conditions. An internal fragment of phiSE14 designated Sdf I has been used as a serovar-specific genetic marker in PCR-based detection systems and as a tool to determine S. Enteritidis levels in experimental infections. The instability of this region may require a reassessment of its suitability for such applications.

FIG. 1.

Schematic representation of φSE14 from S. Enteritidis strain NCTC13349. White arrows represent genes carried in the island, and black arrows represent neighboring genes in the chromosome of NCTC13349. Gray arrows indicate integrase genes. The positions of the DR (attL and attR) flanking the island and of the internal fragment Sdf I are indicated. The locations of primers designed to amplify four fragments of the island by tiling-PCR are shown.

FIG. 2.

The excision of φSE14 generates an extrachromosomal CF of the island. (A) Schematic representation of the excision event showing the locations of primers designed for the detection of the extrachromosomal element. (B) Detection of an extrachromosomal CF of φSE14 in S. Enteritidis strains. The PCR assays were performed as described in Materials and Methods by using primers ISE-Y1(F) and ISE-Y8(R) for external PCR, ISE-Y9(F) and ISE-Y10(R) for inverse PCR, and ISE-Y11(F) and ISE-Y12(R) for nested PCR. The sequence of each primer can be found in Table S2 in the supplemental material. Two independent clones harboring a tetRA-tagged version of φSE14 in the NCTC13349 background (φSE14::tetRA) were included in the analysis.