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

ABSTRACT: The prgB gene encodes aggregation substance (Asc10) which is essential for transfer of the pheromone-inducible conjugative plasmid pCF10 in Enterococcus faecalis. The prgQ and prgS regions, located 4 kb upstream of prgB, are required for the expression of prgB. Complementation studies indicated that the prgQ region functions in cis and in an orientation-dependent manner relative to the prgB gene (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). Analysis of transcriptional fusions in this study, using a promoterless lacZ gene in several locations between prgQ and prgB, confirmed that the prgQ region does not carry a promoter for the expression of prgB and that prgB does not comprise an operon with prgA (which encodes the surface exclusion protein Sec10), the gene immediately upstream from prgB. Northern (RNA) blot analysis demonstrated that two distinct transcripts (Qs RNA and QL RNA), much larger than the prgQ gene, were expressed in the prgQ region. QS RNA was produced constitutively, whereas QL RNA was produced inducibly by pheromone. The lack of any other open reading frame in QL RNA and significant sequence complementarity between the 3' end of QL RNA and the promoter region of prgB suggested that the functional products of the prgQ region might be RNA molecules rather than proteins. A mutation in prgS completely abolished the production of QL RNA. A model for transcriptional activation of prgB is presented.

SUBMITTER: Chung JW

PROVIDER: S-EPMC176856 | biostudies-other | 1995 Apr

REPOSITORIES: biostudies-other

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

Chung J W JW Dunny G M GM

Journal of bacteriology 19950401 8

The prgB gene encodes aggregation substance (Asc10) which is essential for transfer of the pheromone-inducible conjugative plasmid pCF10 in Enterococcus faecalis. The prgQ and prgS regions, located 4 kb upstream of prgB, are required for the expression of prgB. Complementation studies indicated that the prgQ region functions in cis and in an orientation-dependent manner relative to the prgB gene (J. W. Chung and G. M. Dunny, Proc. Natl. Acad. Sci. USA 89:9020-9024, 1992). Analysis of transcripti ...[more]

PMID: 7536732

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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

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

Publications

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

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