bluuebunny/crossref_metadata_2025_split huggingface dataset:
Incorporation of a polypeptide segment into the β‐domain pore during the assembly of a bacterial autotransporter
Authors: Ieva Raffaele , Skillman Kristen M. , Bernstein Harris D. 
Published: August 2010

SummaryBacterial autotransporters consist of an N‐terminal ‘passenger domain’ that is transported into the extracellular space by an unknown mechanism and a C‐terminal ‘β‐domain’ that forms a β‐barrel in the outer membrane. Recent studies have revealed that fully assembled autotransporters have an unusual architecture in which a small passenger domain segment traverses the pore formed by the β‐domain. It is unclear, however, whether this configuration forms prior to passenger domain translocation or results from the translocation of the passenger domain through the β‐domain pore. By examining the accessibility of tobacco etch virus protease sites and single‐cysteine residues in the passenger domain of the Escherichia coli O157:H7 autotransporter EspP at different stages of protein biogenesis, we identified a novel pre‐translocation intermediate whose topology resembles that of the fully assembled protein. This intermediate was isolated in the periplasm in cell fractionation experiments. The data strongly suggest that the EspP β‐domain and an embedded polypeptide segment are integrated into the outer membrane as a single pre‐formed unit. The data also provide indirect evidence that at least some outer membrane proteins acquire considerable tertiary structure prior to their membrane integration.
The Chlamydia outer membrane protein OmcB is required for adhesion and exhibits biovar‐specific differences in glycosaminoglycan binding
Authors: Moelleken Katja , Hegemann Johannes H. 
Published: August 2010

Summary Chlamydia pneumoniae, an obligate intracellular human pathogen, causes a number of respiratory diseases. We explored the role of the conserved OmcB protein in C. pneumoniae infections, using yeast display technology. (i) Yeast cells presenting OmcB were found to adhere to human epithelial cells. (ii) Pre‐incubation of OmcB yeast cells with heparin, but not other glycosaminoglycans (GAGs), abrogated adhesion. (iii) Pre‐treatment of the target cells with heparinase inhibited adherence, and GAG‐deficient CHO cell lines failed to bind OmcB yeast. (iv) A heparin‐binding motif present near the N‐terminus of OmcB is required for host cell binding. (v) Pre‐treatment of chlamydial elementary bodies (EBs) with anti‐OmcB antibody or pre‐incubation of target cells with recombinant OmcB protein reduced infectivity upon challenge with C. pneumoniae. (vi) Adhesion of fluorescently labelled EBs to epithelial or endothelial cells was abrogated by prior addition of heparin or OmcB protein. Thus, C. pneumoniae OmcB is an adhesin that binds heparan sulphate‐like GAGs. OmcB from Chlamydia trachomatis serovar L1 also adheres to human cells in a heparin‐dependent way, unlike its counterpart from serovar E. We show that a single position in the OmcB sequence determines heparin dependence/independence, and variations there may reflect differences between the two serovars in cell tropism and disease pattern.
Undecaprenyl phosphate recycling comes out of age
Authors: Valvano Miguel A. 
Published: August 2010

SummaryThe recycling of the lipid carrier undecaprenyl‐phosphate (Und‐P) requires the dephosphorylation of Und‐PP, a reaction proposed to occur at the external or periplasmic side of the bacterial cell membrane. In this issue of Molecular Microbiology, experiments based on the analysis of lipopolysaccharide modifications in Escherichia coli demonstrate that the phosphorylation of lipid A at position 1 is catalysed by the membrane enzyme LpxT (formerly YeiU). This enzyme specifically transfers the distal phosphate group from Und‐PP to lipid A 1‐phosphate to produce lipid A 1‐diphosphate. Furthermore, this reaction requires a functionally intact MsbA protein, which catalyses the transfer of lipid A across the membrane, confirming that the LpxT‐mediated lipid A modification occurs on the periplasmic side of the membrane. These observations provide a novel and unexpected link between periplasmic lipid A modifications and the Und‐PP recycling pathway.
Division of labour during Bacillus subtilis biofilm formation
Authors: Kearns Daniel B. 
Published: August 2010

SummaryBiofilms are multicellular aggregates of bacteria that are organized by an extracellular matrix. A key determinant of biofilm formation by a variety of bacteria is the proper regulation and assembly of the matrix components. In this issue of Molecular Microbiology, Chai et al. present the surprising finding that the genes encoding the components of the extracellular matrix in Bacillus subtilis are expressed in only a subpopulation of cells. Their results indicate that biofilms contain specialized cell types that are differentiated by transcriptional regulation. The population heterogeneity that they observe is another significant example in a growing list of phenotypes that arise owing to bistable gene expression.
Characterization of five novel Pseudomonas aeruginosa cell‐surface signalling systems
Authors: Llamas María A. , Mooij Marlies J. , Sparrius Marion , Vandenbroucke‐Grauls Christina M. J. E. , Ratledge Colin , Bitter Wilbert 
Published: August 2010

SummaryCell‐surface signalling is a sophisticated regulatory mechanism used by Gram‐negative bacteria to sense signals from outside the cell and transmit them into the cytoplasm. This regulatory system consists of an outer membrane‐localized TonB‐dependent receptor (TonB‐dependent transducer), a cytoplasmic membrane‐localized antisigma factor and an extracytoplasmic function (ECF) sigma factor. Pseudomonas aeruginosa contains 13 potential surface signalling systems of which only six have been studied in detail. In this work we have identified the regulons of five novel P. aeruginosa signalling systems. For that, the ECF sigmas PA0149, PA1912, PA2050, PA2093 and PA4896 have been overexpressed and their target gene candidates have been identified using DNA microarray, proteomic analysis, and/or lacZ reporter construct. All five ECF sigma factors control the production of one TonB‐dependent transducer. Interestingly, two sigma factors, PA2050 and PA2093, regulate the synthesis of a second transducer. Furthermore, we show that although all these sigma factors seem to control putative (metal) transport systems, one of them also regulates the expression of P. aeruginosa pyocins. Finally, we also show that the PA1912‐PA1911‐PA1910 (designated FemI‐FemR‐FemA in this work) signalling system responds to the presence of the Mycobacterium siderophores mycobactin and carboxymycobactin and is involved in the utilization of these heterologous siderophores.
A phage display system designed to detect and study protein–protein interactions
Authors: Bair Catherine L. , Oppenheim Amos , Trostel Andrei , Prag Gali , Adhya Sankar 
Published: August 2010

SummaryAnalysing protein–protein interactions is critical in proteomics and drug discovery. The usage of 2‐Hybrid (2λ) systems is limited to an in vivo environment. We describe a bacteriophage 2‐Hybrid system for studying protein interactions in vitro. Bait and prey are displayed as fusions to the surface of phage λ that are marked with different selectable drug‐resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug‐resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9‐CUE, a Ubiquitin binding domain, and by the interaction of (Gly‐Glu)4 and (Gly‐Arg)4 peptides. Interruptions of the phage interactions by non‐fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a λ‐display library. The unique usefulness to 2λ is also described.
Polymorphism and regulation of the spxB (pyruvate oxidase) virulence factor gene by a CBS‐HotDog domain protein (SpxR) in serotype 2 Streptococcus pneumoniae
Authors: Ramos‐Montañez Smirla , Tsui Ho‐Ching Tiffany , Wayne Kyle J. , Morris Jordan L. , Peters Lindsey E. , Zhang Faming , Kazmierczak Krystyna M. , Sham Lok‐To , Winkler Malcolm E. 
Published: August 2010

Summary spxB‐encoded pyruvate oxidase is a major virulence factor of Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes H2O2 and acetyl phosphate, which play roles in metabolism, signalling, and oxidative stress. We report here the first cis‐ and trans‐acting regulatory elements for spxB transcription. These elements were identified in a genetic screen for spontaneous mutations that caused colonies of strain D39 to change from a semitransparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency ≈ 3 × 10−5) were impaired for SpxB function or expression. Two mutations changed amino acids in SpxB likely required for cofactor or subunit binding. One mutation defined a cis‐acting adjacent direct repeat required for optimal spxB transcription. The other three spontaneous mutations created the same frameshift near the start of the trans‐acting spxR regulatory gene. The SpxR protein contains helix–turn–helix, CBS and HotDog domains implicated in binding DNA, adenosyl compounds, and CoA‐containing compounds respectively, and suggest that SpxR positively regulates spxB transcription in response to energy and metabolic state. Microarray analyses unexpectedly demonstrated that SpxR also positively regulates the strH exoglycosidase gene, which, like spxB, has been implicated in colonization. Finally, SpxR is required for full virulence in a murine model of infection.
Escherichia coli DnaA interacts with HU in initiation at the E. coli replication origin
Authors: Chodavarapu Sundari , Felczak Magdalena M. , Yaniv Josette Rouvière , Kaguni Jon M. 
Published: August 2010

Summary Escherichia coli HU protein is a dimer encoded by two closely related genes whose expression is growth phase‐dependent. As a major component of the bacterial nucleoid, HU binds to DNA non‐specifically, but acts at the chromosomal origin (oriC) during initiation by stimulating strand opening in vitro. We show that the α dimer of HU is more active than other forms of HU in initiation of an oriC‐containing plasmid because it more effectively promotes strand opening of oriC. Other results demonstrate that HU stabilizes the DnaA oligomer bound to oriC, and that the α subunit of HU interacts with the N‐terminal region of DnaA. These observations support a model whereby DnaA interacts with the α dimer or the αβ heterodimer, depending on their cellular abundance, to recruit the respective form of HU to oriC. The greater activity of the α dimer of HU at oriC may stimulate initiation during early log phase compared with the lesser activity of the αβ heterodimer or the β dimer.
The Plasmodium falciparum Maurer's clefts in 3D
Authors: Frischknecht Friedrich , Lanzer Michael 
Published: August 2010

SummaryIn 1902, the German physician Georg Maurer discovered a dotted staining pattern within the cytoplasm of Plasmodium falciparum infected erythrocytes that, according to the tradition at the time, was named in his honour. The significance of Georg Maurer's discovery remained unrecognized for almost a century. Only recently are Maurer's clefts appreciated as a novel type of secretory organelle. Established by the malaria parasite within its host cell, Maurer's clefts play an essential role in directing proteins from the parasite to the erythrocyte surface. In this issue of Molecular Microbiology, Hanssen et al. report on the three dimensional structure of Maurer's clefts, as determined by electron tomography. The data presented suggest that Maurer's clefts are connected to both the parasitophorous vacuolar and the erythrocyte plasma membrane, however, no continuum exists that would allow lipids or proteins to freely flow between these three compartments. This seminal work, which stands in the tradition of Georg Maurer's original discovery, represents a milestone in our understanding of the structure and function of this fascinating organelle.
Trans‐targeting of protease substrates by conformationally activating a regulable ClpX‐recognition motif
Authors: Marshall‐Batty Kimberly R. , Nakai Hiroshi 
Published: August 2010

SummaryConversion of bacteriophage Mu repressor to ClpXP‐sensitive form correlates with induced local flexibility at the ClpX recognition motif located at the C‐terminal end. Changing the C‐terminal valine to an alanine (RepV196A) caused the degradation tag to be constitutively active like that of mutant repressors called Vir, which have a dominant ClpXP‐sensitive conformation. However, unlike Vir, RepV196A was unable to convert wild‐type repressor (Rep) to the ClpXP‐sensitive form. In mixtures with Rep, only RepV196A was rapidly degraded by ClpXP. Unlike Rep, RepV196A was ClpXP sensitive without induced C‐terminal flexibility. And unlike adaptor proteins that tether and deliver substrates to ClpX for trans‐targeting, Vir promoted rapid degradation of Rep by ClpX deleted for the tethering site that binds adaptor proteins. Therefore, Rep's ClpX recognition motif has regulable properties, allowing an alternative trans‐targeting mechanism in which an inactive degradation tag is turned on by induced conformational changes.
Linker histone H1 represses recombination at the ribosomal DNA locus in the budding yeast Saccharomyces cerevisiae
Authors: Li Chonghua , Mueller John E. , Elfline Megan , Bryk Mary 
Published: August 2010

SummarySeveral epigenetic phenomena occur at ribosomal DNA loci in eukaryotic cells, including the silencing of Pol I and Pol II transcribed genes, silencing of replication origins and repression of recombination. In Saccharomyces cerevisiae, studies focusing on the silencing of Pol II transcription and genetic recombination at the ribosomal DNA locus (rDNA) have provided insight into the mechanisms through which chromatin and chromatin‐associated factors regulate gene expression and chromosome stability. The core histones, H2A, H2B, H3 and H4, the fundamental building blocks of chromatin, have been shown to regulate silent chromatin at the rDNA; however, the function of the linker histone H1 has not been well characterized. Here, we show that S. cerevisiae histone H1 represses recombination at the rDNA without affecting Pol II gene silencing. The most highly studied repressor of recombination at the rDNA is the Silent information regulator protein Sir2. We find that cells lacking histone H1 do not exhibit a premature‐ageing phenotype nor do they accumulate the rDNA recombination intermediates and products that are found in cells lacking Sir2. These results suggest that histone H1 represses recombination at the rDNA by a mechanism that is independent of the recombination pathways regulated by Sir2.
Pseudomonas aeruginosa biofilm matrix polysaccharide Psl is regulated transcriptionally by RpoS and post‐transcriptionally by RsmA
Authors: Irie Yasuhiko , Starkey Melissa , Edwards Adrianne N. , Wozniak Daniel J. , Romeo Tony , Parsek Matthew R. 
Published: August 2010

SummaryExtracellular polysaccharides are important components of biofilms. In non‐mucoid Pseudomonas aeruginosa strains, the Pel and Psl polysaccharides are major structural components of the biofilm matrix. In this study, we demonstrate that the alternative σ‐factor RpoS is a positive transcriptional regulator of psl gene expression. Furthermore, we show that psl mRNA has an extensive 5′ untranslated region, to which the post‐transcriptional regulator RsmA binds and represses psl translation. Our observations suggest that upon binding RsmA, the region spanning the ribosome binding site of psl mRNA folds into a secondary stem‐loop structure that blocks the Shine–Dalgarno sequence, preventing ribosome access and protein translation. This constitutes a novel mechanism for translational repression by this family of regulators.
A structural model of anti‐anti‐σ inhibition by a two‐component receiver domain: the PhyR stress response regulator
Authors: Herrou Julien , Foreman Robert , Fiebig Aretha , Crosson Sean 
Published: August 2010

SummaryPhyR is a hybrid stress regulator conserved in α‐proteobacteria that contains an N‐terminal σ‐like (SL) domain and a C‐terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti‐σ factor. PhyR thus functions as an anti‐anti‐σ factor in its phosphorylated state. We present genetic evidence thatCaulobacter crescentusPhyR is a phosphorylation‐dependent stress regulator that functions in the same pathway as σTand its anti‐σ factor, NepR. Additionally, we report the X‐ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti‐anti‐σ regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions σ2and σ4, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of α4‐β5‐α5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions σ2and σ4in the SL domain to open about a flexible connector loop and bind anti‐σ factor.
Differences in longitudinal smooth muscle response along the length of the rabbit small intestine
Authors: SCOTT R. B. , TAN D. T. M. 
Published: August 2010

AbstractTo determine whether there is a gradient in the ability of longitudinal smooth muscle along the length of the small intestine to develop tension we prepared longitudinal muscle strips from proximal (P), medial (M) and distal (D) segments of small intestine removed from 900–1200 g New Zealand white rabbits (n = 9). Isometric tension was recorded in standard tissue baths perfused with oxygenated Krebs solution and 10−6m tetrodotoxin. Basal and active (the response to 10−5m carbachol) length‐tension curves were generated, and the length at which tissues developed maximal active tension (the optimum length or Lo) was determined for P, M and D tissues. Then the dose‐response to carbachol (over the range 10−2‐10−4m) was determined for each of the P, M and D tissues at Lo. Non‐linear regression was used to determine the dose at which the contractile response was half maximal (the ED so), and the maximum tension generated (Tm) by the tissue at each location. The basal tension generated in response to graded stretch increased exponentially between 100 and 140% of initial length and was not significantly different for P, M and D tissues. The active tension generated in response to 10−5m carbachol peaked at 125% of initial length; the Lo for P, M and D tissues was identical. With tissues equilibrated at Lo, dose‐response curves for carbachol and graded concentrations of KCl showed a significant increment in maximal tension generated, but no significant difference in ED50 in the more distal compared to proximal segments. Thus, the passive elastic properties and Lo were identical for the three regions of small intestine examined. However, there was a significant increase in active tension generated in distal compared to more proximal regions of the small intestine, which was not associated with a change in the ED50 value for carbachol. As the response occurs in the presence of tetrodotoxin and can be reproduced with KCl depolarization, it probably reflects post‐receptor changes in smooth muscle function.
Up and down the spinal cord: afferent and efferent innervation of the human external anal sphincter
Authors: ENCK P. , HERDMAN J. , BÖRGERMANN K. , THEISEN U. , ZACCHI P. , LÜBKE H. J. 
Published: August 2010

AbstractAfferent and efferent pathways from the anal canal to the external anal sphincter muscle have been investigated in healthy volunteers by means of scalprecorded somatosensory‐evoked potentials following electrical stimulation within the anal canal and by means of motor‐evoked potentials recorded from the external anal sphincter muscle following transcranial magneto‐electrical stimulation of the motor cortex. Control recordings were obtained following posterior tibial‐nerve stimulation on the one hand and recordings form the anterior tibial muscle on the other hand. Latencies of both somatosensory‐evoked potentials following anal stimulation and motor‐evoked potentials recorded from the external anal sphincter were similar to those of the tibial muscle/nerve despite a longer distance. This may be due to slower conduction of the sensory and motor pathways from and to the pelvic floor in comparison to those pathways innervating the limbs, or to the fact that pelvic floor pathways comprise a larger number of neurons and synapses as has been shown in the motor pathways to the pelvic floor in animals.
Gastrointestinal motor activity associated with postoperative ileus and emesis
Authors: SMITH D. , WALDRON B. , LOUDON M. , SMALL P. , CAMPBELL F. C. 
Published: August 2010

AbstractThe gastrointestinal motor activity associated with post‐operative ileus and emesis has not been fully elucidated. This study has evaluated gastric and small‐bowel motility in six patients before and after cholecystectomy and in six healthy volunteers, by solid‐state manometry. Nausea and vomiting were recorded post‐operatively. After surgery, fasting motor abnormalities including (a) total gastric quiescence and (b) small‐bowel ‘phasic‐bursts’ of contractions were observed in all patients. Phasic bursts (PB) resembled phase III of the migrating motor complex (MMC) on initial visual inspection, but further analysis revealed that they were of shorter duration (3.4 ±.2 min [PB] vs 6.4 ± 0.8 min [MMC], [mean ± SEM] P < 0.01), lower contraction frequency (6.4 ± 0.1 contractions min −1 [PBj vs 10 ± 0.3 contractions min−1 [MMC] [mean ± SEM] P < 0.01) and shorter periodicity (36.4 ± 3 min [PB] vs 70.0 ± 6 min [MMC] [mean ± SEM] P < 0.01). Four patients experienced nausea during phasic burst activity. Vomiting was only observed in association with retrograde phasic‐bursts, which migrated through the duodenum to the stomach. This study has shown consistent gastrointestinal motor abnormalities in the immediate post‐operative state.