Conservation of genomic localization and sequence content of Sau3AI-like restriction-modification gene cassettes among Listeria monocytogenes epidemic clone I and selected strains of serotype 1/2a

Abstract

Listeria monocytogenes is a food-borne pathogen with a clonal population structure and apparently limited gene flow between strains of different lineages. Strains of epidemic clone I (ECI) have been responsible for numerous outbreaks and invariably have DNA that is resistant to digestion by Sau3AI, suggesting methylation of cytosine at GATC sites. A putative restriction-modification (RM) gene cassette has been identified in the genome of the ECI strain F2365 and all other tested ECI strains but is absent from other strains of the same serotype (4b). Homologous RM cassettes have not been reported among L. monocytogenes isolates of other serotypes. Furthermore, conclusive evidence for the involvement of this RM cassette in the Sau3AI resistance phenotype of ECI strains has been lacking. In this study, we describe a highly conserved RM cassette in certain strains of serotypes 1/2a and 4a that have Sau3AI-resistant DNA. In these strains the RM cassette was in the same genomic location as in the ECI reference strain F2365. The cassette included a gene encoding a putative recombinase, suggesting insertion via site-specific recombination. Deletion of the RM cassette in the ECI strain F2365 and the serotype 1/2a strain A7 rendered the DNA of both strains susceptible to Sau3AI digestion, providing conclusive evidence that the cassette includes a gene required for methylation of cytosine at GATC sites in both strains. The findings suggest that, in addition to its presence in ECI strains, this RM cassette and the accompanying genomic DNA methylation is also encountered among selected strains of other lineages.

FIG. 1.

Serotype 1/2a isolates A7 and SE076 with Sau3AI-resistant DNA harbor an RM cassette homologous to that in the ECI strain F2365 and in a genomically equivalent location. PCR primers used were as follows: lanes 1 to 4, strains F2365, A7, SE076, and EGD-e, respectively, with primers 0325F and 0327R, amplifying the putative restriction endonuclease gene and putative methyltransferase gene of F2365; lanes 5 to 8, strains F2365, A7, SE076, and EGD-e, respectively, with primers LMO0321RMdelF and LMO0325R amplifying a fragment spanning a portion of ORFs LMOf2365_0321 and LMOf2365_0325 in F2365; lanes 9 to 12, strains F2365, A7, SE076, and EGD-e, respectively, with primers LMO0328RMdelF and LMO0329RMdelR, amplifying a fragment spanning a portion of ORFs LMOf2365_0329 and LMOf2365_0328 in F2365; lanes 13 to 15, strains EGD-e, A35, and 10, respectively, with primers LMO0321RMdelF and LMO0329RMdelR, flanking the RM cassette of F2365; lane M, DNA size markers (exACTGene cloning DNA ladder; Fisher Scientific). Strains A35 and 10 had Sau3AI-resistant DNA but lacked an RM cassette in this genomic location. PCR was done as described in Materials and Methods and using the primers listed in Table 2.

FIG. 2.

Genomic organization of the region harboring the RM cassette in L. monocytogenes strains F2365 (serotype 4b, ECI), FSL N3-165, A7 (serotype 1/2a), and EGD-e (serotype 1/2a). The arrows indicate the direction of transcription. The pseudogene (LMOf2365_0324 in F2365) is indicated by a stippled arrow. Conserved ORFs on the left and right of the RM cassette are shown as black and gray arrows, respectively. ORFs unique to EGD-e are indicated with horizontal and vertical striations.

FIG. 3.

Evidence for cytosine methylation at GATC sites in wild-type strains and mutants with deletion of the RM cassette. Lanes: 1 to 3, DNA from strain F2365, uncut, digested with Sau3AI, and digested with MboI, respectively; 4 and 5, DNA from deletion mutant DS86 uncut and digested with Sau3AI, respectively; 6 to 8, DNA from strain A7, uncut, digested with Sau3AI, and digested with MboI, respectively; 9 and 10, DNA from deletion mutant DS14 uncut and digested with Sau3AI, respectively; 11 to 13, DNA from FSL N3-165, uncut, digested with Sau3AI, and digested with MboI, respectively; M, molecular weight marker II (Roche Diagnostics, Indianapolis, IN). DNA digestions were as described in Materials and Methods.

FIG. 4.

PFGE fingerprints of serotype 1/2a strains with Sau3AI-resistant and Sau3AI-susceptible DNA. PFGE patterns generated following ApaI digestion were clustered using the BioNumerics program as described in Materials and Methods. RM indicates the presence of the RM cassette in the corresponding isolate with Sau3AI-resistant DNA; ECI-RM indicates isolates with Sau3AI-resistant DNA and harboring an RM cassette homologous to that in the ECI strain F2365 and in the same genomic location; x-RM indicates isolates with Sau3AI-resistant DNA but lacking an RM cassette with homology to that of F2365. Arrows indicate the four distinct PFGE profiles encountered among ECI-RM serotype 1/2a isolates.