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M Tony Hollingsworth,
Gerald W Hart,
James C Paulson,
Elizabeth Stansell,
Kevin Canis,
I-Cheuh Huang,
Maria Panico,
Howard Morris,
Stuart Haslam,
Michael Farzan, [......],
Kazunori Hamamura,
Takenosuke Yoshida,
Kaoru Akita,
Tetsuya Okajima,
Keiko Furukawa,
Takeshi Urano,
Koichi Furukawa,
L Renee Ruhaak,
Suzanne Miyamoto,
Carlito B Lebrilla
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ABSTRACT: Cell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cell surface that enable and block the formation of biologically important molecular complexes. Some mucins, such as MUC1, associate with receptor tyrosine kinases and other cell surface receptors, and engage in signal transduction in order to communicate information regarding conditions at the cell surface to the nucleus. In that context, the MUC1 cytoplasmic tail (MUC1CT) receives phosphorylation signals from receptor tyrosine kinases and serine/threonine kinases, which enables its association with different signaling complexes that conduct these signals to the nucleus and perhaps other subcellular organelles. We have detected the MUC1CT at promoters of over 500 genes, in association with several different transcription factors, and have shown that promoter occupancy can vary under different growth factor conditions. However, the full biochemical nature of the nuclear forms of MUC1 and its function at these promoter regions remain undefined. I will present evidence that nuclear forms of the MUC1CT include extracellular and cytoplasmic tail domains. In addition, I will discuss evidence for a hypothesis that the MUC1CT possesses a novel catalytic function that enables remodeling of the transcription factor occupancy of promoters, and thereby engages in regulation of gene expression.
Glycobiology 11/2011; 21(11):1454-531. · 3.58 Impact Factor
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ABSTRACT: Quorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2.
Differential proteome analysis of wildtype S. Typhimurium versus a luxS mutant revealed relatively few changes beyond the known effect on phase 2 flagellin. However, two highly differentially expressed protein spots with similar molecular weight but differing isoelectric point, were identified as LuxS whereas the S. Typhimurium genome contains only one luxS gene. This observation was further explored and we show that the S. Typhimurium LuxS protein can undergo posttranslational modification at a catalytic cysteine residue. Additionally, by constructing LuxS-betala and LuxS-PhoA fusion proteins, we demonstrate that S. Typhimurium LuxS can substitute the cognate signal peptide sequences of beta-lactamase and alkaline phosphatase for translocation across the cytoplasmic membrane in S. Typhimurium. This was further confirmed by fractionation of S. Typhimurium protein extracts, followed by Western blot analysis.
2D-DIGE analysis of a luxS mutant vs. wildtype Salmonella Typhimurium did not reveal new insights into the role of AI-2/LuxS in Salmonella as only a small amount of proteins were differentially expressed. However, subsequent in depth analysis of the LuxS protein itself revealed two interesting features: posttranslational modification and potential translocation across the cytoplasmic membrane. As the S. Typhimurium LuxS protein does not contain obvious signal motifs, it is speculated that LuxS is a new member of so called moonlighting proteins. These observations might have consequences in future studies on AI-2 quorum signaling in S. Typhimurium.
BMC Microbiology 09/2009; 9:198. · 3.04 Impact Factor
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Karen Lemmens,
Tijl De Bie,
Thomas Dhollander, Sigrid C De Keersmaecker,
Inge M Thijs,
Geert Schoofs,
Ami De Weerdt,
Bart De Moor,
Jos Vanderleyden,
Julio Collado-Vides,
Kristof Engelen,
Kathleen Marchal
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ABSTRACT: We present DISTILLER, a data integration framework for the inference of transcriptional module networks. Experimental validation of predicted targets for the well-studied fumarate nitrate reductase regulator showed the effectiveness of our approach in Escherichia coli. In addition, the condition dependency and modularity of the inferred transcriptional network was studied. Surprisingly, the level of regulatory complexity seemed lower than that which would be expected from RegulonDB, indicating that complex regulatory programs tend to decrease the degree of modularity.
Genome biology 04/2009; 10(3):R27. · 6.63 Impact Factor
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ABSTRACT: The knowledge of molecular mechanisms underlying the adhesive and mechanical properties of cell surface-associated molecules is a key to understanding their functions. In this context, single-molecule force spectroscopy (SMFS) has recently offered new opportunities for probing the adhesion and mechanics of polysaccharides and proteins on live cells. Here we present a protocol that we have used to analyze polysaccharide chains of different nature on the bacterium Lactobacillus rhamnosus GG. We describe procedures (i) for functionalizing atomic force microscopy (AFM) tips with Pseudomonas aeruginosa-I or concanavalin A lectins, (ii) for stretching specific polysaccharide molecules on live bacteria using SMFS with lectin tips and (iii) for mapping the localization, adhesion and extension of individual polysaccharide chains. We also discuss data treatment, emphasizing how to gain insight into the elasticity of the stretched macromolecules using the extended freely jointed chain model. Even though the presented protocol is for L. rhamnosus, it can be easily modified for other cell types. For users having expertise in the field, the entire protocol can be completed in about 5 d.
Nature Protocol 02/2009; 4(6):939-46. · 8.36 Impact Factor
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ABSTRACT: A report of the ESF-EMBO Symposium Bacterial Networks (BacNet08), Sant Feliu de Guixols, Spain, 13-18 September 2008.
Genome biology 12/2008; 9(11):327. · 6.63 Impact Factor
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ABSTRACT: The nanoscale exploration of microbes using atomic force microscopy (AFM) is an exciting, rapidly evolving research field. Here, we show that single-molecule force spectroscopy is a valuable tool for the localization and conformational analysis of individual polysaccharides on live bacteria. We focus on the clinically important probiotic bacterium Lactobacillus rhamnosus GG, demonstrating the power of AFM to reveal the coexistence of polysaccharide chains of different nature on the cell surface. Applicable to a wide variety of cells, this single molecule method offers exciting prospects for analyzing the heterogeneity and diversity of macromolecules constituting cell membranes and cell walls.
ACS Nano 10/2008; 2(9):1921-9. · 10.77 Impact Factor
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ABSTRACT: The PhoPQ two-component system acts as a transcriptional regulator that responds to Mg(2+) starvation both in Escherichia coli and Salmonella typhimurium (Garcia et al. 1996; Kato et al. 1999). By monitoring the availability of extracellular Mg(2+), this two-component system allows S. typhimurium to sense the transition from an extracellular environment to a subcellular location. Concomitantly with this transition, a set of virulence factors essential for survival in the intracellular environment is activated by the PhoPQ system (Groisman et al. 1989; Miller et al. 1989). Compared to nonpathogenic strains, such as E. coli K12, the PhoPQ regulon in pathogens must contain target genes specifically contributing to the virulence phenotype. To verify this hypothesis, we compared the composition of the PhoPQ regulon between E. coli and S. typhimurium using a combination of expression experiments and motif data. PhoPQ-dependent genes in both organisms were identified from PhoPQ-related microarray experiments. To distinguish between direct and indirect targets, we searched for the presence of the regulatory motif in the promoter region of the identified PhoPQ-dependent genes. This allowed us to reconstruct the direct PhoPQ-dependent regulons in E. coli K12 and S. typhimurium LT2. Comparison of both regulons revealed a very limited overlap of PhoPQ-dependent genes between both organisms. These results suggest that the PhoPQ system has acquired a specialized function during evolution in each of these closely related species that allows adaptation to the specificities of their lifestyles (e.g., pathogenesis in S. typhimurium).
Journal of Molecular Evolution 05/2005; 60(4):462-74. · 2.27 Impact Factor
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ABSTRACT: The PmrAB (BasSR) two-component regulatory system is required for Salmonella typhimurium virulence. PmrAB-controlled modifications of the lipopolysaccharide (LPS) layer confer resistance to cationic antibiotic polypeptides, which may allow bacteria to survive within macrophages. The PmrAB system also confers resistance to Fe3+-mediated killing. New targets of the system have recently been discovered that seem not to have a role in the well-described functions of PmrAB, suggesting that the PmrAB-dependent regulon might contain additional, unidentified targets.
We performed an in silico analysis of possible targets of the PmrAB system. Using a motif model of the PmrA binding site in DNA, genome-wide screening was carried out to detect PmrAB target genes. To increase confidence in the predictions, all putative targets were subjected to a cross-species comparison (phylogenetic footprinting) using a Gibbs sampling-based motif-detection procedure. As well as the known targets, we detected additional targets with unknown functions. Four of these were experimentally validated (yibD, aroQ, mig-13 and sseJ). Site-directed mutagenesis of the PmrA-binding site (PmrA box) in yibD revealed specific sequence requirements.
We demonstrated the efficiency of our procedure by recovering most of the known PmrAB-dependent targets and by identifying unknown targets that we were able to validate experimentally. We also pinpointed directions for further research that could help elucidate the S. typhimurium virulence pathway.
Genome biology 02/2004; 5(2):R9. · 6.63 Impact Factor
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ABSTRACT: Introduction The PhoPQ-regulatory system plays an important role in virulence characteristics of Salmonella typhimurium. This two-component system is capable of determining the subcellular location of the organism by sensing Mg and activates virulence factors that allow bacterial survival in an intracellular environment (e.g. macrophages) [2]. Despite the fact that most of the research concerning the PhoPQ-regulatory system has been done on Salmonella spp., an important role of this system is determined in other gamma-proteobacteria such as Escherichia coli, Shigella flexneri and Yersinia pestis: the PhoPQ-activated proteins in these organisms are often related to pathogenicity but also other cellular activities are dedicated to this regulatory system like survival in Mg -limiting conditions. A small DNA-binding site -- (T/G)GTTTA -- has been suggested to be the binding site for the PhoP-regulatory protein in Salmonella spp. [4] and was recently experimentally verified in E. col
07/2003;
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ABSTRACT: Motif detection based on Gibbs sampling is a common procedure used to retrieve regulatory motifs in silico. Using a species-specific background model was previously shown to increase the robustness of the algorithm. Here, we demonstrate that selecting a non-species-adapted background model can have an adverse effect on the results of motif detection. The large differences in the average nucleotide composition of prokaryotic sequences exacerbate the problem of exchanging background models. Therefore, we have developed complex background models for all prokaryotic species with available genome sequences.
Trends in Microbiology 03/2003; 11(2):61-6. · 7.91 Impact Factor
