The ability of Staphylococcus aureus to adapt to various conditions of stress is the result of a complex regulatory response. Previously, it has been demonstrated that Clp homologues are important for a variety of stress conditions, and our laboratory has shown that a clpC homologue was highly expressed in the S. aureus strain DSM20231 during biofilm formation relative to expression in planktonic cells. Persistence and longterm survival are a hallmark of biofilm-associated staphylococcal infections, as cure frequently fails even in the presence of bactericidal antimicrobials. To determine the role of clpC in this context, we performed metabolic, gene expression, and long-term growth and survival analyses of DSM20231 as well as an isogenic clpC allelic-replacement mutant, a sigB mutant, and a clpC sigB double mutant. As expected, the clpC mutant showed increased sensitivity to oxidative and heat stresses. Unanticipated, however, was the reduced expression of the tricarboxylic acid (TCA) cycle gene citB (encoding aconitase), resulting in the loss of aconitase activity and preventing the catabolization of acetate during the stationary phase. clpC inactivation abolished post-stationary-phase recovery but also resulted in significantly enhanced stationary-phase survival compared to that of the wild-type strain. These data demonstrate the critical role of the ClpC ATPase in regulating the TCA cycle and implicate ClpC as being important for recovery from the stationary phase and also for entering the death phase. Understanding the stationary-and post-stationary-phase recovery in S. aureus may have important clinical implications, as little is known about the mechanisms of long-term persistence of chronic S. aureus infections associated with formation of biofilms.

Adherence of Staphylococcus aureus to the host tissue is an important step in the initiation of pathogenesis. At least 10 adhesins produced by S. aureus have been described and it is becoming clear that the expression of these adhesins and their interactions with eukaryotic cells involve complex processes. Some of these, such as the fibronectin-binding proteins (FnBPs) and Clumping Factor A, are well characterized. However, in the last 10 years a number of novel S. aureus adhesins have been described. Functional analyses of these proteins, one of which is Eap (extracellular adherence protein, also known as Map and p70), are revealing important information on the pathogenesis of staphylococcal disease. More than 10 years after the first report of Eap, we are beginning to understand that this protein, which has a broad spectrum of functions, may be a critical factor in the pathogenesis of S. aureus. This review will focus on the interactions of Eap with eukaryotic cells, plasma proteins and the extracellular matrix as well as on the recently recognized role of Eap as an important mediator in the immune response to staphylococcal infection.

Eap and Emp are two Staphylococcus aureus adhesins initially described as extracellular matrix binding proteins. Eap has since emerged as being important in adherence to and invasion of eukaryotic cells, as well as being described as an immunomodulator and virulence factor in chronic infections. This paper describes the mapping of the transcription start point of the eap and emp promoters. Moreover, using reporter-gene assays and real-time PCR in defined regulatory mutants, environmental conditions and global regulators affecting expression of eap and emp were investigated. Marked differences were found in expression of eap and emp between strain Newman and the 8325 derivatives SH1000 and 8325-4. Moreover, both genes were repressed in the presence of glucose. Analysis of expression of both genes in various regulatory mutants revealed that sarA and agr were involved in their regulation, but the data suggested that there were additional regulators of both genes. In a sae mutant, expression of both genes was severely repressed. sae expression was also reduced in the presence of glucose, suggesting that repression of eap and emp in glucose-containing medium may, in part, be a consequence of a decrease in expression of sae. INTRODUCTIONAdhesion of Staphylococcus aureus to eukaryotic cells and implanted devices is an important step in the initiation of staphylococcal infection. Adhesion by S. aureus may be mediated by specific cell-surface proteins, or be a result of interactions of cell-surface proteins with host proteins such as von Willebrand factor, fibronectin, fibrinogen and collagen (Höök & Foster, 2000). By these means, S. aureus can adhere directly to eukaryotic cell receptors or, alternatively, can bind to plasma-coated inserted devices. Navarre & Schneewind, 1999). One set has a characteristic LPXTG motif that anchors the adhesin to the staphylococcal cell surface (Mazmanian et al., 1999). The members of this family of adhesins are called MSCRAMM molecules, and they include protein A, the fibronectin-binding proteins (FnBPs), clumping factors A and B (ClfA and B), and more recently described molecules, such as IsdA, (also known as FrpA and StbA;Wiltshire & Foster, 2001;Mazmanian et al., 2002;Morrissey et al., 2002;Taylor & Heinrichs, 2002), Bsp (Tung et al., 2000) and HarA (Dryla et al., 2003). Many of these proteins have been implicated as bridging molecules between the bacterium and the host cell (e.g. Sinha et al., 1999;Hartleib et al., 2000;Massey et al., 2001). Members of the second set of adhesins are noncovalently anchored to the cell surface, and include the fibrinogen-binding protein (Efb;, coagulase (Boden & Flock, 1989), and Eap and Emp (discussed below). In recent years it has emerged that staphylococcal adhesins may have additional, diverse functions (e.g. Chavakis et al., 2002;Bjerketorp et al., 2004;Heilmann et al., 2005), and that bacteria may alter expression of these molecules in response to changing environmental conditions (e.g. Clarke et al., 2004). Moreover, a number of studies have ...

Matrix attachment regions (MARs) are DNA sequences that may be involved in anchoring DNA/chromatin to the nuclear matrix and they have been described in both mammalian and plant species. MARs possess a number of features that facilitate the opening and maintenance of euchromatin. When incorporated into viral or non-viral vectors MARs can increase transgene expression and limit position-effects. They have been used extensively to improve transgene expression and recombinant protein production and promising studies on the potential use of MAR elements for mammalian gene therapy have appeared. These illustrate how MARs may be used to mediate sustained or higher levels of expression of therapeutic genes and/or to reduce the viral vector multiplicity of infection required to achieve consistent expression. More recently, the discovery of potent MAR elements and the development of improved vectors for transgene delivery, notably non-viral episomal vectors, has strengthened interest in their use to mediate expression of therapeutic transgenes. This article will describe the progress made in this field, and it will discuss future directions and issues to be addressed.

Matrix attachment regions (MAR) generally act as epigenetic regulatory sequences that increase gene expression, and they were proposed to partition chromosomes into loop-forming domains. However, their molecular mode of action remains poorly understood. Here, we assessed the possible contribution of the AT-rich core and adjacent transcription factor binding motifs to the transcription augmenting and anti-silencing effects of human MAR 1–68. Either flanking sequences together with the AT-rich core were required to obtain the full MAR effects. Shortened MAR derivatives retaining full MAR activity were constructed from combinations of the AT-rich sequence and multimerized transcription factor binding motifs, implying that both transcription factors and the AT-rich microsatellite sequence are required to mediate the MAR effect. Genomic analysis indicated that MAR AT-rich cores may be depleted of histones and enriched in RNA polymerase II, providing a molecular interpretation of their chromatin domain insulator and transcriptional augmentation activities.