AraC-Type Regulator Rbf Controls the Staphylococcus epidermidis Biofilm Phenotype by Negatively Regulating the icaADBC Repressor SarR.

Regulation of icaADBC-encoded polysaccharide intercellular adhesin (PIA)/poly-N-acetylglucosasmine (PNAG) production in staphylococci plays an important role in biofilm-associated medical-device-related infections. Here, we report that the AraC-type transcriptional regulator Rbf activates icaADBC operon transcription and PIA production in Staphylococcus epidermidis Purified recombinant Rbf did not bind to the ica operon promoter region in electrophoretic mobility shift assays (EMSAs), indicating that Rbf regulates ica transcription indirectly. To identify the putative transcription factor(s) involved in Rbf-mediated icaADBC regulation, the ability of recombinant Rbf to interact with the promoter sequences of known icaADBC regulators was investigated. Recombinant Rbf bound to the sarR promoter and not the sarX, sarA, sarZ, spx, and srrA promoters. Reverse transcription (RT)-PCR demonstrated that Rbf acts as a repressor of sarR transcription. PIA expression and biofilm production were restored to wild-type levels in an rbf sarR double mutant grown in brain heart infusion (BHI) medium supplemented with NaCl, which is known to activate the ica locus, but not in BHI medium alone. RT-PCR further demonstrated that although Rbf does not bind the sarX promoter, it nevertheless exerted a negative effect on sarX expression. Apparently, direct downregulation of the SarR repressor by Rbf has a dominant effect over indirect repression of the SarX activator by Rbf in the control of S. epidermidis PIA production and biofilm formation. IMPORTANCE: The importance of Staphylococcus epidermidis as an opportunistic pathogen in hospital patients with implanted medical devices derives largely from its capacity to form biofilm. Expression of the icaADBC-encoded extracellular polysaccharide is the predominant biofilm mechanism in S. epidermidis clinical isolates and is tightly regulated. Here, we report that the transcriptional regulator Rbf promotes icaADBC expression by negatively regulating expression of sarR, which encodes an ica operon repressor. Furthermore, Rbf indirectly represses the ica operon activator, SarX. The data reveal complicated interplay between Rbf and two Sar family proteins in fine-tuning regulation of the biofilm phenotype and indicate that in the hierarchy of biofilm regulators, IcaR is dominant over the Rbf-SarR-SarX axis.

Evaluation of the role of the vaccinia virus uracil DNA glycosylase and A20 proteins as intrinsic components of the DNA polymerase holoenzyme.

The vaccinia virus DNA polymerase is inherently distributive but acquires processivity by associating with a heterodimeric processivity factor comprised of the viral A20 and D4 proteins. D4 is also an enzymatically active uracil DNA glycosylase (UDG). The presence of an active repair protein as an essential component of the polymerase holoenzyme is a unique feature of the replication machinery. We have shown previously that the A20-UDG complex has a stoichiometry of ∼1:1, and our data suggest that A20 serves as a bridge between polymerase and UDG. Here we show that conserved hydrophobic residues in the N' terminus of A20 are important for its binding to UDG. Our data argue against the assembly of D4 into higher order multimers, suggesting that the processivity factor does not form a toroidal ring around the DNA. Instead, we hypothesize that the intrinsic, processive DNA scanning activity of UDG tethers the holoenzyme to the DNA template. The inclusion of UDG as an essential holoenzyme component suggests that replication and base excision repair may be coupled. Here we show that the DNA polymerase can utilize dUTP as a substrate in vitro. Moreover, uracil moieties incorporated into the nascent strand during holoenzyme-mediated DNA synthesis can be excised by the viral UDG present within this holoenzyme, leaving abasic sites. Finally, we show that the polymerase stalls upon encountering an abasic site in the template strand, indicating that, like many replicative polymerases, the poxviral holoenzyme cannot perform translesion synthesis across an abasic site.

Mice deficient in the serine/threonine protein kinase VRK1 are infertile due to a progressive loss of spermatogonia.

The VRK1 protein kinase has been implicated as a pro-proliferative factor. Genetic analyses of mutant alleles of the Drosophila and Caenorhabditis elegans VRK1 homologs have revealed phenotypes ranging from embryonic lethality to mitotic and meiotic defects with resultant sterility. Herein, we describe the first genetic analysis of murine VRK1. Two lines of mice containing distinct gene-trap integrations into the Vrk1 locus were established. Insertion into intron 12 (GT12) spared VRK1 function, enabling the examination of VRK1 expression in situ. Insertion into intron 3 (GT3) disrupted VRK1 function, but incomplete splicing to the gene trap rendered this allele hypomorphic (approximately 15% of wild-type levels of VRK1 remain). GT3/GT3 mice are viable, but both males and females are infertile. In testes, VRK1 is expressed in Sertoli cells and spermatogonia. The infertility of GT3/GT3 male mice results from a progressive defect in spermatogonial proliferation or differentiation, culminating in the absence of mitotic and meiotic cells in adult testis. These data demonstrate an important role for VRK1 in cell proliferation and confirm that the need for VRK1 during gametogenesis is evolutionarily conserved.

Isolation of chromosomal and plasmid DNA from Staphylococcus epidermidis.

The following describes noncommercial methods for the purification of genomic and plasmid DNA from S. epidermidis. These include both large-scale, high molecular weight and quick, small-scale chromosomal DNA extractions, and also a standard alkaline lysis method of plasmid preparation.

A dysfunctional tricarboxylic acid cycle enhances fitness of Staphylococcus epidermidis during ß-lactam stress.

UNLABELLED: A recent controversial hypothesis suggested that the bactericidal action of antibiotics is due to the generation of endogenous reactive oxygen species (ROS), a process requiring the citric acid cycle (tricarboxylic acid [TCA] cycle). To test this hypothesis, we assessed the ability of oxacillin to induce ROS production and cell death in Staphylococcus epidermidis strain 1457 and an isogenic citric acid cycle mutant. Our results confirm a contributory role for TCA-dependent ROS in enhancing susceptibility of S. epidermidis toward ß-lactam antibiotics and also revealed a propensity for clinical isolates to accumulate TCA cycle dysfunctions presumably as a way to tolerate these antibiotics. The increased protection from ß-lactam antibiotics could result from pleiotropic effects of a dysfunctional TCA cycle, including increased resistance to oxidative stress, reduced susceptibility to autolysis, and a more positively charged cell surface. IMPORTANCE: Staphylococcus epidermidis, a normal inhabitant of the human skin microflora, is the most common cause of indwelling medical device infections. In the present study, we analyzed 126 clinical S. epidermidis isolates and discovered that tricarboxylic acid (TCA) cycle dysfunctions are relatively common in the clinical environment. We determined that a dysfunctional TCA cycle enables S. epidermidis to resist oxidative stress and alter its cell surface properties, making it less susceptible to ß-lactam antibiotics.