Physical and genetic map of Enterococcus faecium ATCC19434 and demonstration of intra- and interspecific genomic diversity in enterococci

Abstract

A physical map of the Enterococcus faecium ATCC19434 chromosome was constructed by NotI, I-CeuI and Sse8387I. The chromosome was a circular DNA of 2600 kb in size, and contained six rRNA operons (rrn). The locations and orientations of the six rrn operons and 24 different determinants were mapped. Genomes of three additional E. faecium strains were also analyzed by I-CeuI digestion, and the genome sizes were found to vary from 2550 to 2995 kb. We further investigated the genome sizes and number of rrn operons in four E. faecalis, one E. avium, and one E. durans strains. The genome sizes were larger than E. faecium: 3000^3250 kb in E. faecalis, 3445 kb in E. avium, and 3070 kb in E. durans. E. avium and E. durans contained six rrn operons as in E. faecium, but all the E. faecalis strains possessed four rrn operons. ß

Putative Surface Proteins Encoded within a Novel Transferable Locus Confer a High-Biofilm Phenotype to Enterococcus faecalis

“…Filter mating experiments, however, revealed that this locus is able to transfer to recipient E. faecalis strains at high frequencies, typical for conjugative plasmids. To determine the nature of the genetic element harboring the bee locus, we employed a CHEF gel approach previously used to examine genome diversity in enterococci (22) and to assign a chromosomal location to the vanG operon in E. faecalis (1). The enzyme I-ceuI is an intron-encoded endonuclease that specifically recognizes unique sequences present within rrn operons (22).…”

“…To determine the nature of the genetic element harboring the bee locus, we employed a CHEF gel approach previously used to examine genome diversity in enterococci (22) and to assign a chromosomal location to the vanG operon in E. faecalis (1). The enzyme I-ceuI is an intron-encoded endonuclease that specifically recognizes unique sequences present within rrn operons (22). E. faecalis strains were postulated to contain four rrn operons based on mapping experiments (22), and this was subsequently confirmed by the complete genome sequence of strain V583 (25).…”

“…The enzyme I-ceuI is an intron-encoded endonuclease that specifically recognizes unique sequences present within rrn operons (22). E. faecalis strains were postulated to contain four rrn operons based on mapping experiments (22), and this was subsequently confirmed by the complete genome sequence of strain V583 (25). CHEF gel analysis of I-ceuI-digested DNA from E. faecalis followed by Southern hybridization analysis with 23S rRNA gene probes would therefore be expected to identify the four chromosomal fragments.…”

“…CHEF gel analysis was performed as described previously (1,22,40) with some modifications. A portion of the agarose plug containing total DNA from each strain was cut with a sterile razor and restricted with I-ceuI (New England Biolabs, Beverly, MA) at 37°C for 16 h. The plugs were washed in 1 ml dilute Tris-EDTA for 1 h at 37°C, then carefully loaded into the wells of a 1% pulsed-field certified agarose gel (Bio-Rad Laboratories, Hercules, CA) in 0.5ϫ TBE (45 mM Tris HCl, 45 mM boric acid, 1 mM EDTA), and electrophoresed using a CHEF DRII device (Bio-Rad Laboratories, Hercules, CA) with the pulse time ramped from 5 to 120 s at 150 V for 40 h. The gels were then stained with ethidium bromide and photographed using a UVP gel documentation system (UVP Inc., Upland, CA) before the DNA was transferred to Zeta-probe GT genomic-tested blotting membrane by capillary or vacuum blotting.…”

Comparison of Genotypic and Phylogenetic Relationships of Environmental Enterococcus Isolates by BOX-PCR Typing and 16S rRNA Gene Sequencing

“…BOX-PCR typing targets sequences located between interspersed repetitive DNA elements, resulting in amplification products of different sizes that generate a genomic fingerprint of individual bacterial strains. Variation in genome sizes (26), as well as the location of BOX elements among different strains of a particular Enterococcus species, leads to generation of multiple strain-specific fingerprint patterns, which allows BOX-PCR typing to discriminate among different strains of the same species better than 16S rRNA gene sequencing.…”