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Genetic analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions.

ABSTRACT: The prgB gene encodes the surface protein Asc10, which mediates cell aggregation resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline resistance plasmid pCF10 in Enterococcus faecalis. Previous Tn5 insertional mutagenesis and sequencing analysis of a 12-kb fragment of pCF10 indicated that a region containing prgX, -Q, -R, -S, and -T, located 3 to 6 kb upstream of prgB, is required to activate the expression of prgB. Complementation studies showed that the positive regulatory region functions in cis in an orientation-dependent manner (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). In order to determine the involvement of each gene in the activation of prgB, Tn5 insertional mutagenesis and exonuclease III deletion analyses of the regulatory region were carried out. The results indicate that prgQ and -S are required for the expression of prgB, while prgX, -R, and -T are not required. Western blot (immunoblot) analysis of these mutants shows that prgQ is also essential for the expression of prgA (encoding the surface exclusion protein Sec10), which is located between prgB and the positive-control region. Complementation analysis demonstrates that a cis-acting regulatory element is located in the prgQ region and that pCF10 sequences in an untranslated region 3' from prgQ are an essential component of the positive-control system. Analyses of various Tn5 insertions in pCF10 genes suggest that transcription reading into this transposon is terminated in E. faecalis but that outward-reading transcripts may initiate from within the ends of Tn5 or from the junction sequences.

SUBMITTER: Chung JW

PROVIDER: S-EPMC176855 | biostudies-other | 1995 Apr

REPOSITORIES: biostudies-other

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Genetic analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions.

Chung J W JW Bensing B A BA Dunny G M GM

Journal of bacteriology 19950401 8

The prgB gene encodes the surface protein Asc10, which mediates cell aggregation resulting in high-frequency conjugative transfer of the pheromone-inducible tetracycline resistance plasmid pCF10 in Enterococcus faecalis. Previous Tn5 insertional mutagenesis and sequencing analysis of a 12-kb fragment of pCF10 indicated that a region containing prgX, -Q, -R, -S, and -T, located 3 to 6 kb upstream of prgB, is required to activate the expression of prgB. Complementation studies showed that the posi ...[more]

PMID: 7721703

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Project description:Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E. faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E. faecalis. Here, we explore the hypothesis that multidrug-resistant E. faecalis strains are immunocompromised. We assessed MGE acquisition by E. faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620 kb of horizontally acquired genome content that characterizes V583. T11 possesses the E. faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E. faecalis CRISPR1-cas locus, which most multidrug-resistant E. faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E. faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E. faecalis. IMPORTANCE Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics.

Project description: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.

Dataset Information

Genetic analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions.

Publications

Genetic analysis of a region of the Enterococcus faecalis plasmid pCF10 involved in positive regulation of conjugative transfer functions.

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