Biosynthesis of selenate reductase in <i>Salmonella enterica</i>:critical roles for the signal peptide and DmsD

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium with a flexible respiratory capability. Under anaerobic conditions, S. enterica can utilize a range of terminal electron acceptors, including selenate, to sustain respiratory electron transport. The S. enterica selenate reductase is a membrane-bound enzyme encoded by the ynfEFGH-dmsD operon. The active enzyme is predicted to comprise at least three subunits where YnfE is a molybdenum-containing catalytic subunit. The YnfE protein is synthesized with an N-terminal twin-arginine signal peptide and biosynthesis of the enzyme is coordinated by a signal peptide binding chaperone called DmsD. In this work, the interaction between S. enterica DmsD and the YnfE signal peptide has been studied by chemical crosslinking. These experiments were complemented by genetic approaches, which identified the DmsD binding epitope within the YnfE signal peptide. YnfE signal peptide residues L24 and A28 were shown to be important for assembly of an active selenate reductase. Conversely, a random genetic screen identified the DmsD V16 residue as being important for signal peptide recognition and selenate reductase assembly

A membrane-depolarizing toxin substrate of the Staphylococcus aureus type VII secretion system mediates intraspecies competition.

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date, only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane-depolarizing activity is alleviated by coproduction of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologs at the tspA locus, suggestive of additional roles in intraspecies competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologs

Design and validation of the Health Professionals' Attitudes Toward the Homeless Inventory (HPATHI)

BACKGROUND: Recent literature has called for humanistic care of patients and for medical schools to begin incorporating humanism into medical education. To assess the attitudes of health-care professionals toward homeless patients and to demonstrate how those attitudes might impact optimal care, we developed and validated a new survey instrument, the Health Professional Attitudes Toward the Homeless Inventory (HPATHI). An instrument that measures providers' attitudes toward the homeless could offer meaningful information for the design and implementation of educational activities that foster more compassionate homeless health care. Our intention was to describe the process of designing and validating the new instrument and to discuss the usefulness of the instrument for assessing the impact of educational experiences that involve working directly with the homeless on the attitudes, interest, and confidence of medical students and other health-care professionals. METHODS: The study consisted of three phases: identifying items for the instrument; pilot testing the initial instrument with a group of 72 third-year medical students; and modifying and administering the instrument in its revised form to 160 health-care professionals and third-year medical students. The instrument was analyzed for reliability and validity throughout the process. RESULTS: A 19-item version of the HPATHI had good internal consistency with a Cronbach's alpha of 0.88 and a test-retest reliability coefficient of 0.69. The HPATHI showed good concurrent validity, and respondents with more than one year of experience with homeless patients scored significantly higher than did those with less experience. Factor analysis yielded three subscales: Personal Advocacy, Social Advocacy, and Cynicism. CONCLUSIONS: The HPATHI demonstrated strong reliability for the total scale and satisfactory test-retest reliability. Extreme group comparisons suggested that experience with the homeless rather than medical training itself could affect health-care professionals' attitudes toward the homeless. This could have implications for the evaluation of medical school curricula

The type VII secretion system of <i>Staphylococcus aureus</i> secretes a nuclease toxin that targets competitor bacteria

The type VII protein secretion system (T7SS) plays a critical role in the virulence of human pathogens including Mycobacterium tuberculosis and Staphylococcus aureus. Here we report that the S. aureus T7SS secretes a large nuclease toxin, EsaD. The toxic activity of EsaD is neutralised during its biosynthesis through complex formation with an antitoxin, EsaG, which binds to its C-terminal nuclease domain. The secretion of EsaD is dependent upon a further accessory protein, EsaE, that does not interact with the nuclease domain, but instead binds to the EsaD N-terminal region. EsaE has a dual cytoplasmic/membrane localization and membrane-bound EsaE interacts with the T7SS secretion ATPase, EssC, implicating EsaE in targeting the EsaDG complex to the secretion apparatus. EsaD and EsaE are co-secreted whereas EsaG is found only in the cytoplasm and may be stripped off during the secretion process. Strain variants of S. aureus that lack esaD encode at least two copies of EsaG-like proteins most likely to protect themselves from the toxic activity of EsaD secreted by esaD(+) strains. In support of this, a strain overproducing EsaD elicits significant growth inhibition against a sensitive strain. We conclude that T7SSs may play unexpected and key roles in bacterial competitiveness

Hoare Logic-based Genetic Programming

Almost all existing genetic programming systems deal with fitness evaluation solely by testing. In this paper, by contrast, we present an original approach that combines genetic programming with Hoare logic with the aid of model checking and finite state automata, henceby proposing a brand new verification-focused formal genetic programming system that makes it possible to evolve reliable programs with mathematically-verified properties

Velocity-space sensitivity of the time-of-flight neutron spectrometer at JET

The velocity-space sensitivities of fast-ion diagnostics are often described by so-called weight functions. Recently, we formulated weight functions showing the velocity-space sensitivity of the often dominant beam-target part of neutron energy spectra. These weight functions for neutron emission spectrometry (NES) are independent of the particular NES diagnostic. Here we apply these NES weight functions to the time-of-flight spectrometer TOFOR at JET. By taking the instrumental response function of TOFOR into account, we calculate time-of-flight NES weight functions that enable us to directly determine the velocity-space sensitivity of a given part of a measured time-of-flight spectrum from TOFOR

Relationship of edge localized mode burst times with divertor flux loop signal phase in JET

A phase relationship is identified between sequential edge localized modes (ELMs) occurrence times in a set of H-mode tokamak plasmas to the voltage measured in full flux azimuthal loops in the divertor region. We focus on plasmas in the Joint European Torus where a steady H-mode is sustained over several seconds, during which ELMs are observed in the Be II emission at the divertor. The ELMs analysed arise from intrinsic ELMing, in that there is no deliberate intent to control the ELMing process by external means. We use ELM timings derived from the Be II signal to perform direct time domain analysis of the full flux loop VLD2 and VLD3 signals, which provide a high cadence global measurement proportional to the voltage induced by changes in poloidal magnetic flux. Specifically, we examine how the time interval between pairs of successive ELMs is linked to the time-evolving phase of the full flux loop signals. Each ELM produces a clear early pulse in the full flux loop signals, whose peak time is used to condition our analysis. The arrival time of the following ELM, relative to this pulse, is found to fall into one of two categories: (i) prompt ELMs, which are directly paced by the initial response seen in the flux loop signals; and (ii) all other ELMs, which occur after the initial response of the full flux loop signals has decayed in amplitude. The times at which ELMs in category (ii) occur, relative to the first ELM of the pair, are clustered at times when the instantaneous phase of the full flux loop signal is close to its value at the time of the first ELM

Transport of Folded Proteins by the Tat System

The twin-arginine protein translocation (Tat) system has been characterized in bacteria, archaea and the chloroplast thylakoidal membrane. This system is distinct from other protein transport systems with respect to two key features. Firstly, it accepts cargo proteins with an N-terminal signal peptide that carries the canonical twin-arginine motif, which is essential for transport. Second, the Tat system only accepts and translocates fully folded cargo proteins across the respective membrane. Here, we review the core essential features of folded protein transport via the bacterial Tat system, using the three-component TatABC system of Escherichia coli and the two-component TatAC systems of Bacillus subtilis as the main examples. In particular, we address features of twin-arginine signal peptides, the essential Tat components and how they assemble into different complexes, mechanistic features and energetics of Tat-dependent protein translocation, cytoplasmic chaperoning of Tat cargo proteins, and the remarkable proofreading capabilities of the Tat system. In doing so, we present the current state of our understanding of Tat-dependent protein translocation across biological membranes, which may serve as a lead for future investigations