N-Terminal Region of CusB Is Sufficient for Metal Binding and Metal Transfer with the Metallochaperone CusF

Gram-negative bacteria, such as Escherichia coli, utilize efflux resistance systems in order to expel toxins from their cells. Heavy-metal resistance is mediated by resistance nodulation cell division (RND)-based efflux pumps composed of a tripartite complex that includes an RND-transporter, an outer-membrane factor (OMF), and a membrane fusion protein (MFP) that spans the periplasmic space. MFPs are necessary for complex assembly and have been hypothesized to play an active role in substrate efflux. Crystal structures of MFPs are available, however incomplete, as large portions of the apparently disordered N- and C-termini are unresolved. Such is the case for CusB, the MFP of the E. coli Cu(I)/Ag(I) efflux pump CusCFBA. In this work, we have investigated the structure and function of the N-terminal region of CusB, which includes the metal-binding site and is missing from previously determined crystal structures. Results from mass spectrometry and X-ray absorption spectroscopy show that the isolated N-terminal 61 residues (CusB-NT) bind metal in a 1:1 stoichiometry with a coordination site composed of M21, M36, and M38, consistent with full-length CusB. NMR spectra show that CusB-NT is mostly disordered in the apo state; however, some slight structure is adopted upon metal binding. Much of the intact protein's function is maintained in this fragment as CusB-NT binds metal in vivo and in vitro, and metal is transferred between the metallochaperone CusF and CusB-NT in vitro. Functional analysis in vivo shows that full-length CusB is necessary in an intact polypeptide for full metal resistance, though CusB-NT alone can contribute partial metal resistance. These findings reinforce the theory that the role of CusB is not only to bind metal but also to play an active role in efflux.     

Evidence of inbreeding depression on human height

Stature is a classical and highly heritable complex trait, with 80%-90% of variation explained by genetic factors. In recent years, genome-wide association studies (GWAS) have successfully identified many common additive variants influencing human height; however, little attention has been given to the potential role of recessive genetic effects. Here, we investigated genome-wide recessive effects by an analysis of inbreeding depression on adult height in over 35,000 people from 21 different population samples. We found a highly significant inverse association between height and genome-wide homozygosity, equivalent to a height reduction of up to 3 cm in the offspring of first cousins compared with the offspring of unrelated individuals, an effect which remained after controlling for the effects of socio-economic status, an important confounder (χ(2) = 83.89, df = 1; p = 5.2 × 10(-20)). There was, however, a high degree of heterogeneity among populations: whereas the direction of the effect was consistent across most population samples, the effect size differed significantly among populations. It is likely that this reflects true biological heterogeneity: whether or not an effect can be observed will depend on both the variance in homozygosity in the population and the chance inheritance of individual recessive genotypes. These results predict that multiple, rare, recessive variants influence human height. Although this exploratory work focuses on height alone, the methodology developed is generally applicable to heritable quantitative traits (QT), paving the way for an investigation into inbreeding effects, and therefore genetic architecture, on a range of QT of biomedical importance.     

Associations between Purine Metabolites and Clinical Symptoms in Schizophrenia

Background

The antioxidant defense system, which is known to be dysregulated in schizophrenia, is closely linked to the dynamics of purine pathway. Thus, alterations in the homeostatic balance in the purine pathway may be involved in the pathophysiology of schizophrenia.

Methodology/Principal Findings

Breakdown products in purine pathway were measured using high-pressure liquid chromatography coupled with a coulometric multi-electrode array system for 25 first-episode neuroleptic-naïve patients with schizophrenia at baseline and at 4-weeks following initiation of treatment with antipsychotic medication. Associations between these metabolites and clinical and neurological symptoms were examined at both time points. The ratio of uric acid and guanine measured at baseline predicted clinical improvement following four weeks of treatment with antipsychotic medication. Baseline levels of purine metabolites also predicted clinical and neurological symtpoms recorded at baseline; level of guanosine was associated with degree of clinical thought disturbance, and the ratio of xanthosine to guanosine at baseline predicted degree of impairment in the repetition and sequencing of actions.

Conclusions/Significance

Findings suggest an association between optimal levels of purine byproducts and dynamics in clinical symptoms and adjustment, as well as in the integrity of sensory and motor processing. Taken together, alterations in purine catabolism may have clinical relevance in schizophrenia pathology.     

Evidence that Cryptosporidium parvum Populations Are Panmictic and Unstructured in the Upper Midwest of the United States

Cryptosporidium parvum is a zoonotic protozoan parasite that causes cryptosporidiosis, an infectious diarrheal disease primarily affecting humans and neonatal ruminants. Understanding the transmission dynamics of C. parvum, particularly the specific contributions of zoonotic and anthroponotic transmission, is critical to the control of this pathogen. This study used a population genetics approach to better understand the transmission of C. parvum in the Upper Midwest United States. A total of 254 C. parvum isolates from cases of human cryptosporidiosis in Minnesota and Wisconsin and diarrheic calves in Minnesota, Wisconsin, and North Dakota were genotyped at eight polymorphic loci. Isolates with a complete profile from all eight loci (n = 212) were used to derive a multilocus genotype (MLT), which was used in population genetic analyses. Among the 94 MLTs identified, 60 were represented by a single isolate. Approximately 20% of isolates belonged to MLT 2, a group that included both human and cattle isolates. Population analyses revealed a predominantly panmictic population with no apparent geographic or host substructuring.     

Regulation of Cu(I)/Ag(I) efflux genes in Escherichia coli by the sensor kinase CusS

Two-component systems are widely used by bacteria to mediate adaptive responses to a variety of environmental stimuli. The CusR/CusS two-component system in Escherichia coli induces expression of genes involved in metal efflux under conditions of elevated Cu(I) and Ag(I) concentrations. As seen in most prototypical two-component systems, signal recognition and transmission is expected to occur by ligand binding in the periplasmic sensor domain of the histidine kinase CusS. Although discussed in the extant literature, little experimental evidence is available to establish the role of CusS in metal homeostasis. In this study, we show that the cusS gene is required for Cu(I) and Ag(I) resistance in E.?coli and that CusS is linked to the expression of the cusCFBA genes. These results show a metal-dependent mechanism of CusS activation and suggest an absolute requirement for CusS in Cu(I)- and Ag(I)-dependent upregulation of cusCFBA expression in E.?coli.     

A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes

Regulated antisense RNA (asRNA) expression has been employed successfully in Gram-positive bacteria for genome-wide essential gene identification and drug target determination. However, there have been no published reports describing the application of asRNA gene silencing for comprehensive analyses of essential genes in Gram-negative bacteria. In this study, we report the first genome-wide identification of asRNA constructs for essential genes in Escherichia coli. We screened 250?000 library transformants for conditional growth inhibitory recombinant clones from two shotgun genomic libraries of E.?coli using a paired-termini expression vector (pHN678). After sequencing plasmid inserts of 675 confirmed inducer sensitive cell clones, we identified 152 separate asRNA constructs of which 134 inserts came from essential genes, while 18 originated from nonessential genes (but share operons with essential genes). Among the 79 individual essential genes silenced by these asRNA constructs, 61 genes (77%) engage in processes related to protein synthesis. The cell-based assays of an asRNA clone targeting fusA (encoding elongation factor G) showed that the induced cells were sensitized 12-fold to fusidic acid, a known specific inhibitor. Our results demonstrate the utility of the paired-termini expression vector and feasibility of large-scale gene silencing in E.?coli using regulated asRNA expression.     

The histidine kinase CusS senses silver ions through direct binding by its sensor domain

The Cus system of Escherichia coli aids in protection of cells from high concentrations of Ag(I) and Cu(I). The histidine kinase CusS of the CusRS two-component system functions as a Ag(I)/Cu(I)-responsive sensor kinase and is essential for induction of the genes encoding the CusCFBA efflux pump. In this study, we have examined the molecular features of the sensor domain of CusS in order to understand how a metal-responsive histidine kinase senses specific metal ions. We find that the predicted periplasmic sensor domain of CusS directly interacts with Ag(I) ions and undergoes a conformational change upon metal binding. Metal binding also enhances the tendency of the domain to dimerize. These findings suggest a model for activation of the histidine kinase through metal binding events in the periplasmic sensor domain.