A novel conjugative plasmid from Enterococcus faecalis E99 enhances resistance to ultraviolet radiation

Abstract

Enterococcus faecalis has emerged as a prominent healthcare-associated pathogen frequently encountered in bacteremia, endocarditis, urinary tract infection, and as a leading cause of antibiotic-resistant infections. We recently demonstrated a capacity for high-level biofilm formation by a clinical E. faecalis isolate, E99. This high biofilm-forming phenotype was attributable to a novel locus, designated bee, specifying a pilus at the bacterial cell surface and localized to a large approximately 80 kb conjugative plasmid. To better understand the origin of the bee locus, as well as to potentially identify additional factors important to the biology and pathogenesis of strain E99, we sequenced the entire plasmid. The nucleotide sequence of the plasmid, designated pBEE99, revealed large regions of identity to the previously characterized conjugative plasmid pCF10. In addition to the bee locus, pBEE99 possesses an open reading frame potentially encoding aggregation substance, as well as open reading frames putatively encoding polypeptides with 60% to 99% identity at the amino acid level to proteins involved in regulation of the pheromone response and conjugal transfer of pCF10. However, strain E99 did not respond to the cCF10 pheromone in clumping assays. While pBEE99 was found to be devoid of any readily recognizable antibiotic resistance determinants, it carries two non-identical impB/mucB/samB-type genes, as well as genes potentially encoding a two-component bacteriocin similar to that encoded on pYI14. Although no bacteriocin activity was detected from an OG1RF transconjugant carrying pBEE99 against strain FA2-2, it was approximately an order of magnitude more resistant to ultraviolet radiation. Moreover, curing strain E99 of this plasmid significantly reduced its ability to survive UV exposure. Therefore, pBEE99 represents a novel conjugative plasmid that confers biofilm-forming and enhanced UV resistance traits that might potentially impact the virulence and/or fitness of E. faecalis.

Fig. 1

Physical map of plasmid pBEE99 showing the putative ORFs and their orientations. The molecular size of pBEE99 was determined to be 80,600 base pairs and 95 ORFs were identified using the FGENESB software. Putative genes are labeled with the closest match (lowest E value) from protein BLAST searches. Genes in which no hits were found are labeled with the ORF number (column 1 from Table 1). Pseudo-genes or interrupted ORFs are labeled with the original gene name and an asterisk.

Fig. 2

Comparison of the UV resistance of strain OG1RF to that of strain IG9 (an OG1RF transconjugant possessing pBEE99). IG9 demonstrated approximately an order of magnitude greater UV resistance than the parental strain OG1RF. Shown is a representative of two independent experiments.

Fig. 3

Plasmid pBEE99 specifies enhanced resistance to ultraviolet radiation. Strain E99 demonstrated significantly greater resistance than the pBEE99-cured strain KDS01 (P = 0.038), and strain IG9 was significantly more resistant to UV than was the parental strain OG1RF (P = 0.048). Experiments were conducted in triplicate and the error bars indicate the mean ± standard deviation. Levels of significance were determined using a Student’s t-test.