Enterococcus faecalis (EF) is both a common commensal of the human gastrointestinal tract (GI) and a leading cause of hospital acquired infections1. Systemic infections with multi-drug resistant enterococci occur subsequent to GI colonization2. Preventing colonization by multi-drug resistant EF could therefore be a valuable approach to limiting infection. However, little is known about mechanisms EF uses to colonize and compete for stable gastrointestinal niches. Pheromone-responsive, conjugative plasmids encoding bacteriocins are common among enterococcal strains3, and could modulate niche competition among enterococci or between enterococci and the intestinal microbiota. We developed a model of mouse gut colonization with EF without disrupting the microbiota, to evaluate the role of the conjugative plasmid pPD1 expressing bacteriocin 214 on enterococcal colonization. Here we show that EF harboring pPD1 replaces indigenous enterococci and outcompetes EF lacking pPD1. Furthermore, in the intestine, pPD1 is transferred to other EF strains by conjugation, enhancing their survival. Moreover, colonization with an EF strain carrying a conjugation-defective pPD1 mutant resulted in clearance of vancomycin-resistant enterococci, without plasmid transfer. Therefore bacteriocin expression by commensal bacteria can influence niche-competition in the GI tract, and bacteriocins, delivered by commensals that occupy a precise intestinal bacterial niche, may be an effective therapeutic approach to specifically eliminate intestinal colonization by multi-drug resistant bacteria, without profound disruption of the indigenous microbiota.

The global transcriptional regulator Spx of Bacillus subtilis is controlled at several levels of the gene expression process. It is maintained at low concentrations during unperturbed growth by the ATP-dependent protease ClpXP. Under disulfide stress, Spx concentration increases due in part to a reduction in ClpXPcatalyzed proteolysis. Recent studies of Larsson and coworkers (Mol. Microbiol. 66:669-684, 2007) implicated the product of the yjbH gene as being necessary for the proteolytic control of Spx. In the present study, yeast two-hybrid analysis and protein-protein cross-linking showed that Spx interacts with YjbH. YjbH protein was shown to enhance the proteolysis of Spx in reaction mixtures containing ClpXP protease but not ClpCP protease. An N-terminal truncated form of YjbH with a deletion of residues 1 to 24 (YjbH ⌬1-24 ) showed no proteolysis enhancement activity. YjbH is specific for Spx as it did not accelerate proteolysis of the ClpXP substrate green fluorescent protein (GFP)-SsrA, a GFP derivative with a C-terminal SsrA tag that is recognized by ClpXP. Using inductively coupled plasma atomic emission spectroscopy and 4-(2-pyridylazo) resorcinol release experiments, YjbH was found to contain zinc atoms. Zinc analysis of YjbH ⌬1-24 revealed that the N-terminal histidine-rich region is indispensable for the coordination of at least one Zn atom. A Zn atom coordinated by the N-terminal region was rapidly released from the protein upon treatment with a strong oxidant. In conclusion, YjbH is proposed to be an adaptor for ClpXP-catalyzed Spx degradation, and a model of YjbH redox control involving Zn dissociation is presented.In the spore-forming bacterium Bacillus subtilis, the protein Spx is a global transcriptional regulator that exerts both positive and negative control of multiple genes during thiol-specific oxidative stress (34, 57). Cells undergoing disulfide stress exhibit an elevated Spx concentration and heightened Spx activity. This leads to induction of the Spx regulon, which includes trxA (encoding thioredoxin), trxB (thioredoxin reductase), and other genes that function in the oxidative stress response and in cysteine biosynthesis (10, 34). Spx-dependent activation of trxA and trxB requires interaction of the protein with RNA polymerase (RNAP) holoenzyme (33,37). This interaction also results in repression of operons that require an activator for transcription initiation (35, 54).The spx gene was first identified as a suppressor locus of clpP and clpX mutations (32). ClpX proteins form hexameric, ringshaped complexes and belong to the AAAϩ (for ATPases associated with a variety of cellular activities) Clp/Hsp100 family of proteins (2). ClpX is the ATPase and substrate-binding component of the ClpXP protease. ClpX also functions as an unfoldase and translocase that can denature and deliver substrate proteins to the proteolytic chamber, which is composed of two heptameric rings of the ClpP subunits (45). In wild-type cells under normal growth conditions, Spx is present at nearly undetectable l...

Summary NsrR is a nitric oxide (NO)-

Vancomycin‐resistant Enterococcus (VRE) poses a serious threat in hospitals where they densely colonize the intestinal tracts of patients. In vulnerable hosts, these pathogens may translocate to the bloodstream and become lethal. The ability to selectively reduce VRE in the intestinal tracts of patients could potentially prevent many of these translocation events and reduce the spread of the pathogen. Herein, we have engineered Escherichia. coli Nissle 1917 to produce and secrete three antimicrobial peptides, Enterocin A, Enterocin B, and Hiracin JM79, to specifically target and kill Enterococcus. These peptides exhibited potent activity against both Enterococcus faecium and Enterococcus faecalis, the two most prominent species responsible for VRE infections. We first discuss the optimization of the system used to express and secrete the peptides. We then show that by simultaneously expressing these peptides, both E. faecium and E. faecalis were drastically inhibited. We then demonstrate a suppression of the development of resistance when supernatant from the E. coli producer strains was used to treat E. faecium. Finally, we tested the efficacy of the probiotic in a VRE colonization model in mice. These studies showed that administration of the engineered probiotic significantly reduced the levels of both E. faecium and E. faecalis in the feces of male Balb/cJ mice.