Sustained pupil responses are modulated by predictability of auditory sequences

The brain is highly sensitive to auditory regularities and exploits the predictable order of sounds in many situations, from parsing complex auditory scenes, to the acquisition of language. To understand the impact of stimulus predictability on perception, it is important to determine how the detection of predictable structure influences processing and attention. Here we use pupillometry to gain insight into the effect of sensory regularity on arousal. Pupillometry is a commonly used measure of salience and processing effort, with more perceptually salient or perceptually demanding stimuli consistently associated with larger pupil diameters.In two experiments we tracked human listeners' pupil dynamics while they listened to sequences of 50ms tone pips of different frequencies. The order of the tone pips was either random, contained deterministic (fully predictable) regularities (experiment 1, n = 18, 11 female) or had a probabilistic regularity structure (experiment 2, n = 20, 17 female). The sequences were rapid, preventing conscious tracking of sequence structure thus allowing us to focus on the automatic extraction of different types of regularities. We hypothesized that if regularity facilitates processing by reducing processing demands, a smaller pupil diameter would be seen in response to regular relative to random patterns. Conversely, if regularity is associated with heightened arousal and attention (i.e. engages processing resources) the opposite pattern would be expected. In both experiments we observed a smaller sustained (tonic) pupil diameter for regular compared with random sequences, consistent with the former hypothesis and confirming that predictability facilitates sequence processing.SIGNIFICANCE STATEMENTThe brain is highly sensitive to auditory regularities. To appreciate the impact that the presence of predictability has on perception, we need to better understand how a predictable structure influences processing and attention. We recorded listeners' pupil responses to sequences of tones that followed either a predictable or unpredictable pattern, as the pupil can be used to implicitly tap into these different cognitive processes. We found that the pupil showed a smaller sustained diameter to predictable sequences, indicating that predictability eased processing rather than boosted attention. The findings suggest that the pupil response can be used to study the automatic extraction of regularities, and that the effects are most consistent with predictability helping the listener to efficiently process upcoming sounds

Structure of HrcQ(B)-C, a conserved component of the bacterial type III secretion systems

Type III secretion systems enable plant and animal bacterial pathogens to deliver virulence proteins into the cytosol of eukaryotic host cells, causing a broad spectrum of diseases including bacteremia, septicemia, typhoid fever, and bubonic plague in mammals, and localized lesions, systemic wilting, and blights in plants. In addition, type III secretion systems are also required for biogenesis of the bacterial flagellum. The HrcQ(B) protein, a component of the secretion apparatus of Pseudomonas syringae with homologues in all type III systems, has a variable N-terminal and a conserved C-terminal domain (HrcQ(B)-C). Here, we report the crystal structure of HrcQ(B)-C and show that this domain retains the ability of the full-length protein to interact with other type III components. A 3D analysis of sequence conservation patterns reveals two clusters of residues potentially involved in protein–protein interactions. Based on the analogies between HrcQ(B) and its flagellum homologues, we propose that HrcQ(B)-C participates in the formation of a C-ring-like assembly

Defining the Rhizobium leguminosarum Species Complex

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a ‘natural’ unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, “R. indicum” and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains

Defining the Rhizobium leguminosarum Species Complex

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a ‘natural’ unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, “R. indicum” and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains

Bacterial Delivery of Nuclear Proteins into Pluripotent and Differentiated Cells

Numerous Gram negative pathogens possess a type III secretion system (T3SS) which allows them to inject virulent proteins directly into the eukaryotic cell cytoplasm. Injection of these proteins is dependent on a variable secretion signal sequence. In this study, we utilized the N-terminal secretion signal sequence of Pseudomonas aeruginosa exotoxin ExoS to translocate Cre recombinase containing a nuclear localization sequence (Cre-NLS). Transient exposure of human sarcoma cell line, containing Cre-dependent lacZ reporter, resulted in efficient recombination in the host chromosome, indicating that the bacterially delivered protein was not only efficiently localized to the nucleus but also retained its biological function. Using this system, we also illustrate the ability of P. aeruginosa to infect mouse embryonic stem cells (mESC) and the susceptibility of these cells to bacterially delivered Cre-NLS. A single two-hour infection caused as high as 30% of the mESC reporter cells to undergo loxP mediated chromosomal DNA recombination. A simple antibiotic treatment completely eliminated the bacterial cells following the delivery, while the use of an engineered mutant strain greatly reduced cytotoxicity. Utility of the system was demonstrated by delivery of the Cre-NLS to induced pluripotent stem cells to excise the floxed oncogenic nuclear reprogramming cassette. These results validate the use of T3SS for the delivery of transcription factors for the purpose of cellular reprogramming

Cell Membrane Is Impaired, Accompanied by Enhanced Type III Secretion System Expression in Yersinia pestis Deficient in RovA Regulator

BACKGROUND: In the enteropathogenic Yersinia species, RovA regulates the expression of invasin, which is important for enteropathogenic pathogenesis but is inactivated in Yersinia pestis. Investigation of the RovA regulon in Y. pestis at 26 °C has revealed that RovA is a global regulator that contributes to virulence in part by the direct regulation of psaEFABC. However, the regulatory roles of RovA in Y. pestis at 37 °C, which allows most virulence factors in mammalian hosts to be expressed, are still poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: The transcriptional profile of an in-frame rovA mutant of Y. pestis biovar Microtus strain 201 was analyzed under type III secretion system (T3SS) induction conditions using microarray techniques, and it was revealed that many cell-envelope and transport/binding proteins were differentially expressed in the ΔrovA mutant. Most noticeably, many of the T3SS genes, including operons encoding the translocon, needle and Yop (Yersinia outer protein) effectors, were significantly up-regulated. Analysis of Yop proteins confirmed that YopE and YopJ were also expressed in greater amounts in the mutant. However, electrophoresis mobility shift assay results demonstrated that the His-RovA protein could not bind to the promoter sequences of the T3SS genes, suggesting that an indirect regulatory mechanism is involved. Transmission electron microscopy analysis indicated that there are small loose electron dense particle-like structures that surround the outer membrane of the mutant cells. The bacterial membrane permeability to CFSE (carboxyfluorescein diacetate succinimidyl ester) was significantly decreased in the ΔrovA mutant compared to the wild-type strain. Taken together, these results revealed the improper construction and dysfunction of the membrane in the ΔrovA mutant. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the RovA regulator plays critical roles in the construction and functioning of the bacterial membrane, which sheds considerable light on the regulatory functions of RovA in antibiotic resistance and environmental adaptation. The expression of T3SS was upregulated in the ΔrovA mutant through an indirect regulatory mechanism, which is possibly related to the altered membrane construction in the mutant

Type III protein translocase - Hrcn is a peripheral membrane ATPase that is activated by oligomerization

Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F-1/V-1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa ( monomer); II, similar to300 kDa ( putative hexamer); III, 575 kDa ( dodecamer); and IV, similar to3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (> 700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane

HrpZ(Psph) from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro

The hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. Some hrp gene products constitute elements of the type III secretion system, by which effector proteins are exported and delivered into plant cells. Here, we show that the hrpZ gene product from the bean halo-blight pathogen, Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)), is secreted in an hrp-dependent manner in P. syringae pv. phaseolicola and exported by the type III secretion system in the mammalian pathogen Yersinia enterocolitica. HrpZ(Psph) was found to associate stably with liposomes and synthetic bilayer membranes. Under symmetric ionic conditions, addition of 2 nM of purified recombinant HrpZ(Psph) to the cis compartment of planar lipid bilayers provoked an ion current with a large unitary conductivity of 207 pS. HrpZ(Psph)-related proteins from P. syringae pv. tomato or syringae triggered ion currents similar to those stimulated by HrpZ(Psph). The HrpZ(Psph)-mediated ion-conducting pore was permeable for cations but did not mediate fluxes of Cl-. Such pore-forming activity may allow nutrient release and/or delivery of virulence factors during bacterial colonization of host plants