BcrR has been identified as a novel regulatory protein of high level bacitracin resistance encoded by the bcrABD operon in Enterococcus faecalis. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNAbinding proteins, and topological modeling predicts that the C-terminal domain contains four transmembrane ␣-helices. These data have led to the hypothesis that BcrR functions as both a membrane-bound sensor and transducer of bacitracin availability to regulate bcrABD expression. To characterize the bcrABD promoter and identify the promoter elements to which BcrR binds, a series of bcrA-lacZ fusions were constructed. A 69-bp region was identified that was essential for bacitracin-dependent bcrA-lacZ expression. Mutations that targeted this region were used to identify two inverted repeat sequences, each with the sequence 5-GACA(N) 7 TGTC-3, on the bcrABD promoter that were required for bcrA-lacZ expression. To study BcrR binding to this region, we overproduced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-␤-D-maltoside, and subsequently purified it via Ni 2؉ -nitrilotriacetic acid and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes, and BcrR binding to bcrABD promoter DNA was analyzed using electrophoretic mobility shift assays. Both inverted repeat sequences were required for BcrR binding, both in the presence and absence of bacitracin. These data demonstrate that membrane-bound BcrR binds specifically to the bcrABD promoter, irrespective of bacitracin concentration. We therefore propose that bacitracin-dependent induction of bcrABD expression by BcrR occurs after DNA binding.Bacitracin is a polypeptide antibiotic that functions by binding to and sequestering undecaprenyl pyrophosphate (UPP) 4 (1). Because UPP acts as a carrier of peptidoglycan monomeric units across the cell membrane (2), the antibacterial nature of bacitracin results from its ability to block cell wall synthesis. High level bacitracin resistance in Enterococcus faecalis is encoded by the bcrABD operon that is under the control of a putative membrane-bound DNA-binding protein, BcrR (3). In the presence of bacitracin, transcription of the bcrABD operon leads to the production of BcrA and BcrB, which are proposed to act as an ABC exporter of bacitracin, thus conferring high level bacitracin resistance (minimum inhibitory concentration Ն 256 g/ml) to the cell (3). BcrD is proposed to function as an undecaprenyl pyrophosphate phosphatase that functions to increase the amount of undecaprenyl available to the cell (3). It has been demonstrated that bcrR, which is transcribed constitutively, is essential for high level bacitracin resistance in E. faecalis and that transcription of bcrABD is abolished in the absence of bcrR (3). The expression of bcrABD was found to be inducible with increasing concentrations of bacitracin (3). The N-terminal domain (amino acid residues 5-61) of BcrR has homology to the Xre famil...

In the model organism E. coli, recombination mediated by the related XerC and XerD recombinases complexed with the FtsK translocase at specialized dif sites, resolves dimeric chromosomes into free monomers to allow efficient chromosome segregation at cell division. Computational genome analysis of Helicobacter pylori, a slow growing gastric pathogen, identified just one chromosomal xer gene (xerH) and its cognate dif site (difH). Here we show that recombination between directly repeated difH sites requires XerH, FtsK but not XerT, the TnPZ transposon associated recombinase. xerH inactivation was not lethal, but resulted in increased DNA per cell, suggesting defective chromosome segregation. The xerH mutant also failed to colonize mice, and was more susceptible to UV and ciprofloxacin, which induce DNA breakage, and thereby recombination and chromosome dimer formation. xerH inactivation and overexpression each led to a DNA segregation defect, suggesting a role for Xer recombination in regulation of replication. In addition to chromosome dimer resolution and based on the absence of genes for topoisomerase IV (parC, parE) in H. pylori, we speculate that XerH may contribute to chromosome decatenation, although possible involvement of H. pylori's DNA gyrase and topoisomerase III homologue are also considered. Further analyses of this system should contribute to general understanding of and possibly therapy development for H. pylori, which causes peptic ulcers and gastric cancer; for the closely related, diarrheagenic Campylobacter species; and for unrelated slow growing pathogens that lack topoisomerase IV, such as Mycobacterium tuberculosis.

Several lines of study suggest that peripheral metabolism of amyloid beta (Aß) is associated with risk for Alzheimer disease (AD). In blood, greater than 90% of Aß is complexed as an apolipoprotein, raising the possibility of a lipoprotein-mediated axis for AD risk. In this study, we report that genetic modification of C57BL/6J mice engineered to synthesise human Aß only in liver (hepatocyte-specific human amyloid (HSHA) strain) has marked neurodegeneration concomitant with capillary dysfunction, parenchymal extravasation of lipoprotein-Aß, and neurovascular inflammation. Moreover, the HSHA mice showed impaired performance in the passive avoidance test, suggesting impairment in hippocampal-dependent learning. Transmission electron microscopy shows marked neurovascular disruption in HSHA mice. This study provides causal evidence of a lipoprotein-Aß /capillary axis for onset and progression of a neurodegenerative process.

CbrA is an atypical sensor kinase found in Pseudomonas. The autokinase domain is connected to a putative transporter of the sodium/solute symporter family (SSSF). CbrA functions together with its cognate response regulator, CbrB, and plays an important role in nutrient acquisition, including regulation of hut genes for the utilization of histidine and its derivative, urocanate. Here we report on the findings of a genetic and biochemical analysis of CbrA with a focus on the function of the putative transporter domain. The work was initiated with mutagenesis of histidine uptake-proficient strains to identify histidine-specific transport genes located outside the hut operon. Genes encoding transporters were not identified, but mutations were repeatedly found in cbrA. This, coupled with the findings of [ 3 H]histidine transport assays and further mutagenesis, implicated CbrA in histidine uptake. In addition, mutations in different regions of the SSSF domain abolished signal transduction. Site-specific mutations were made at four conserved residues: W55 and G172 (SSSF domain), H766 (H box), and N876 (N box). The mutations W55G, G172H, and N876G compromised histidine transport but had minimal effects on signal transduction. The H766G mutation abolished both transport and signal transduction, but the capacity to transport histidine was restored upon complementation with a transport-defective allele of CbrA, most likely due to interdomain interactions. Our combined data implicate the SSSF domain of CbrA in histidine transport and suggest that transport is coupled to signal transduction. IMPORTANCENutrient acquisition in bacteria typically involves membrane-bound sensors that, via cognate response regulators, determine the activity of specific transporters. However, nutrient perception and uptake are often coupled processes. Thus, from a physiological perspective, it would make sense for systems that couple the process of signaling and transport within a single protein and where transport is itself the stimulus that precipitates signal transduction to have evolved. The CbrA regulator in Pseudomonas represents a unique type of sensor kinase whose autokinase domain is connected to a transporter domain. We present genetic and biochemical evidence that suggests that CbrA plays a dual role in histidine uptake and sensing and that transport is dependent on signal transduction.T he ability to recognize and convert external environmental stimuli into appropriate physiological responses is of fundamental importance for all organisms. In bacteria, signal transduction is predominantly mediated by two-component regulatory systems (TCSs) consisting of a sensor kinase (SK) and a cognate response regulator (RR) (1, 2). Both proteins are typically composed of two distinct functional domains (3): a variable N-terminal signal input domain and a conserved C-terminal autokinase domain for the SK and a conserved N-terminal receiver (Rec) domain and a variable C-terminal output domain for the RR. Signal transduction is achieved via ph...

BackgroundOne mechanism utilized by bacterial pathogens for host adaptation and immune evasion is the generation of phenotypic diversity by the phasevarion that results from the differential expression of a suite of genes regulated by the activity of a phase-variable methyltransferase within a restriction modification (RM) system. Phasevarions are active in Helicobacter pylori, however there have been no studies investigating the significance of phase-variable RM systems on host colonization.MethodsTwo mutant types incapable of phase variation were constructed; a clean deletion mutant (‘DEL’) and a mutant (‘ON’) where the homopolymeric repeat was replaced with a non-repeat synonymous sequence, resulting in expression of the full-length protein. The resulting mutants were assessed for their colonisation ability in the mouse model.ResultsFive phase-variable genes encoding either methyltransferases or members of RM systems were found in H. pylori OND79. Our mutants fell into three categories; 1, those with little effect on colonization, 2, those where expression of the full-length protein was detrimental, 3, those where both mutations were detrimental.ConclusionsOur results demonstrated that phase-variable methyltransferases are critical to H. pylori colonization, suggesting that genome methylation and generation of epigenetic diversity is important for colonization and pathogenesis. The third category of mutants suggests that differential genome methylation status of H. pylori cell populations, achieved by the phasevarion, is essential for host adaptation. Studies of phase-variable RM mutants falling in the two other categories, not strictly required for colonization, represent a future perspective to investigate the role of phasevarion in persistence of H. pylori.