Influence of laterally acquired genetic elements on the physiology of Salmonella enterica

INFLUENCE OF LATERALLY ACQUIRED GENETIC ELEMENTS ON THE PHYSIOLOGY OF SALMONELLA ENTERICA Salmonella spp. are accountable for a large fraction of the global infectious disease burden, with most of their infections being food- or water-borne. As the phenotypic features and adaptive potential of Salmonella spp. appear to be driven mainly by mobile or laterally acquired genetic elements, a better understanding of the behaviour and diversification of these important pathogens consequently requires a more profound insight into the different mechanisms by which these pivotal elements can affect cellular physiology. In this work, research was accordingly focused on the impact of a laterally acquired endonuclease (i.e. Mrr), and a temperate bacteriophage (i.e. P22) on the physiology of Salmonella Typhimurium LT2. Mrr is a cryptic Type IV restriction endonuclease that is characterized by its specificity for modified DNA. The gene encoding Mrr is located in the immigration control region , a foreign genetic element that harbours a large number of restriction systems. In contrast to the Mrr protein of its close relative, Escherichia coli MG1655, we initially found Mrr of LT2 to be inactive. Closer analysis, however, revealed that degeneration of LT2 Mrr might have been enforced by the presence of a laterally acquired Type III methyltransferase in LT2, whose activity in the cell is incompatible with a functional Mrr enzyme. Nevertheless, random mutagenesis revealed that a number of seemingly unrelated mutations could each still render LT2 Mrr constitutively active, indicating its degeneration is readily reversible. P22, on the other hand, is a temperate and Salmonella-specific model phage that over the last 50 years has greatly contributed to our understanding of phage biology and phage host interactions in general. Using a random promoter-trap library, we stumbled upon a novel interaction between P22 and LT2, which is characterized by the deliberate redirection of the host s metabolism. Interestingly, we could identify the actual instigator of this interaction as a small ORFan protein encoded on the P22 genome, but its deletion did not obviously affect infection or lysogenisation by P22. In summary, the research in this work revealed a number of novel mechanisms by which specific mobile or laterally acquired genetic elements can determine or interfere with the behaviour of S. Typhimurium.status: publishe

Evaluation of spatial interpolation methods for annual rainfall on the highlands of Eritrea

No Abstract. Discovery and Innovation Vol. 18(1) 2006: 15-2

Phage–host interactions during pseudolysogeny: Lessons from the Pid/dgo interaction

Although the study of phage infection has a long history and catalyzed much of our current understanding in bacterial genetics, molecular biology, evolution and ecology, it seems that microbiologists have only just begun to explore the intricacy of phage-host interactions. In a recent manuscript by Cenens et al. we found molecular and genetic support for pseudolysogenic development in the Salmonella Typhimurium-phage P22 model system. More specifically, we observed the existence of phage carrier cells harboring an episomal P22 element that segregated asymmetrically upon subsequent divisions. Moreover, a newly discovered P22 ORFan protein (Pid) able to derepress a metabolic operon of the host (dgo) proved to be specifically expressed in these phage carrier cells. In this addendum we expand on our view regarding pseudolysogeny and its effects on bacterial and phage biology.status: publishe

Flagellin hypervariable region determines symbiotic properties of commensal Escherichia coli strains.

Escherichia coli represents a classical intestinal gram-negative commensal. Despite this commensalism, different E. coli strains can mediate disparate immunogenic properties in a given host. Symbiotic E. coli strains such as E. coli Nissle 1917 (EcN) are attributed beneficial properties, e.g., promotion of intestinal homeostasis. Therefore, we aimed to identify molecular features derived from symbiotic bacteria that might help to develop innovative therapeutic alternatives for the treatment of intestinal immune disorders. This study was performed using the dextran sodium sulphate (DSS)-induced colitis mouse model, which is routinely used to evaluate potential therapeutics for the treatment of Inflammatory Bowel Diseases (IBDs). We focused on the analysis of flagellin structures of different E. coli strains. EcN flagellin was found to harbor a substantially longer hypervariable region (HVR) compared to other commensal E. coli strains, and this longer HVR mediated symbiotic properties through stronger activation of Toll-like receptor (TLR)5, thereby resulting in interleukin (IL)-22-mediated protection of mice against DSS-induced colitis. Furthermore, using bone-marrow-chimeric mice (BMCM), CD11c+ cells of the colonic lamina propria (LP) were identified as the main mediators of these flagellin-induced symbiotic effects. We propose flagellin from symbiotic E. coli strains as a potential therapeutic to restore intestinal immune homeostasis, e.g., for the treatment of IBD patients

Isolation and stoichiometry analysis of the SpaPR subcomplex of the needle complex.

<p>(A) Elution profile of the purified SpaPR^FLAG complex run on a Superdex 200 10/300 GL column. The peaks corresponding to the SpaPR^FLAG complex and 3xFLAG peptide are indicated. (B) Coomassie-stained SDS PAGE gel of purified SpaPR^FLAG complex and of its FLAG-deficient control (left). Immunodetection of SpaP (green) and SpaR^FLAG (red) on Western blot from purified SpaPR^FLAG complex separated by SDS PAGE (right). (C) Traces of indicated detector signals from size exclusion chromatography—multi angle laser light scattering of purified SpaPR^FLAG complex (left). ASTRA-calculated mass profile of total components of peak of purified SpaPR^FLAG complex (polypeptides and detergent, middle). ASTRA-calculated mass profile polypeptide components of peak of purified SpaPR^FLAG complex (right). (D) Native mass spectrum of the SpaPR^STREP complex. Peak series corresponding to the SpaP:SpaR^STREP complex in a 5:1 ratio is marked in red, with the most abundant charge state (14+) indicated. The peak series marked in blue corresponds to the same SpaPR complex bound to a ligand with a mass of approximately 710 Da, indicative of an associated phospholipid. Note that the measured mass for SpaPR heterohexamer (157.882 kDa) is heavier than the theoretically calculated mass (157.280 kDa). Abbreviations: Coo: Coomassie stained, WB: Western blot, RI: refractive index, LS: light scattering.</p

SpaP-SpaP interactions analyzed by <i>in vivo</i> photocrosslinking and sequence co-variation.

<p>(A) Immunodetection of SpaP^FLAG on Western blots of crude membrane samples of <i>E</i>. <i>coli</i> BL21 (DE3) expressing SpaP_T15X^FLAG in the absence of all other T3SS components. The sample is shown with and without UV-irradiation to induce photocrosslinking of <i>p</i>Bpa to neighboring interaction partners. (B) Immunodetection of chromosome-encoded SpaP^FLAG on Western blots of crude membrane samples of <i>S</i>. Typhimurium expressing plasmid-encoded SpaP_T15X. (C) Immunodetection of SpaP^FLAG and the inner MS ring protein PrgK on Western blots of crude membrane samples of <i>S</i>. Typhimurium expressing indicated SpaP-<i>p</i>Bpa mutants separated by 2-dimensional blue native/SDS PAGE. Full 2D gels are only shown for SpaP^FLAG scanned in the 800 nm channel. The 2D gel showing SpaP_M187X^FLAG +UV has been re-probed with antibody for PrgK and scanned in the 700 nm channel. PrgK indicates the position of the assembled needle complex. An overlay of FLAG and PrgK signals is shown on the right. The relevant slice of the 700 nm image showing PrgK at 25 kDa and the overlay of both channels showing the needle complex-associated bands have been aligned to the corresponding 2D image. (D) Interaction map of SpaP. Lines indicate predicted interactions with a normalized coupling score > 0.8 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006071#ppat.1006071.s003" target="_blank">S3 Table</a>) at positions with experimentally identified SpaP-SpaP crosslinks (at least from one side). Positions with experimentally observed SpaP-SpaP interactions are shown in black, target positions only predicted are shown in light blue. Grey shading indicates TM helices. Only positions within or in close proximity to TM helices are shown. Abbreviations: chr—chromosomal.</p

Interactions among the export apparatus components SpaP, SpaQ, SpaR, and SpaS.

<p>(A) Immunodetection of SpaR^FLAG on Western blots of SDS PAGE-separated crude membrane samples of Δ<i>spaPQRS S</i>. Typhimurium expressing indicated SpaP-<i>p</i>Bpa mutants from a pT10-<i>spaPQR</i>^FLAG<i>S</i> plasmid. (B) Immunodetection of SpaP^FLAG on Western blots of SDS PAGE-separated crude membrane samples of Δ<i>spaPQRS S</i>. Typhimurium expressing indicated SpaR-<i>p</i>Bpa mutants from a pT10-<i>spaP</i>^FLAG<i>QRS</i> plasmid. (C) Immunodetection of SpaS_N258A^FLAG on Western blots of SDS PAGE-separated crude membrane samples of <i>S</i>. Typhimurium expressing indicated plasmid-complemented SpaP-<i>p</i>Bpa mutants. (D) As in (C) but assessing the SpaP-SpaS interaction in absence of the inner ring protein PrgK. (E) Immunodetection of SpaP^FLAG on Western blots of SDS PAGE-separated crude membrane samples of <i>S</i>. Typhimurium expressing chromosome-encoded indicated SpaP-<i>p</i>Bpa mutants in the presence or absence of the inner ring protein PrgK. (F) As in (E) but showing SpaR_M209X^FLAG. (G) Immunodetection of SpaP^FLAG on Western blots of crude membrane samples of <i>E</i>. <i>coli</i> BL21 (DE3) expressing indicated SpaP-<i>p</i>Bpa mutants together with SpaQRS to form the SpaPR complex. (H) As in (F) but expressing SpaP_V170XQR^FLAGS to reveal the SpaP-SpaR interaction in <i>E</i>. <i>coli</i>.</p

Visualization and characterization of the pore formed by SpaP and SpaR.

<p>(A) Six selected class averages (4, 23, 29, 36, 55, 82) of negative-stained isolated SpaPR complexes imaged by electron microscopy. The length of the scale bar represents 50 Å. The two class averages at the top represent the SpaP₅ complex. Arrowheads in the class averages in the middle and at the bottom represent the anticipated position of SpaR on the SpaP₅ ring. The complete picture of all class averages can be seen in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006071#ppat.1006071.s009" target="_blank">S4 Fig</a>. (B) Fluorescent streptavidin detection of SDS PAGE-separated biotin maleimide-labeled proteins of whole cell lysates, cell culture supernatant, periplasmic fraction, or cytoplasmic fraction of <i>S</i>. Typhimurium Δ<i>prgHIJK</i>, <i>flhD</i>::<i>tet</i> moderately overexpressing indicated proteins from a medium copy number plasmid (pT12). (C) Blue native PAGE and immunodetection of a high molecular weight complex formed by EPEA-tagged SpaP alone.</p